Optimized mini-dystrophin genes and expression cassettes and their use

ABSTRACT

This invention relates to polynucleotides encoding mini-dystrophin proteins, viral vectors comprising the same, and methods of using the same for delivery of mini-dystrophin to a cell or a subject.

STATEMENT OF PRIORITY

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 16/787,938, filed on Feb. 11, 2020, now U.S. Pat.No. 11,547,765, which is a continuation of and claims priority to U.S.patent application Ser. No. 15/628,268, filed on Jun. 20, 2017, nowabandoned, which claims the benefit of U.S. Provisional Application Ser.No. 62/352,675, filed on Jun. 21, 2016, and U.S. Provisional ApplicationSer. No. 62/516,449, filed on Jun. 7, 2017, the contents of each ofwhich are incorporated herein by reference in their entirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in XML format, submitted under 37 C.F.R. §1.831-1.834, entitled 5470-784CT2_ST26.xml, 80,246 bytes in size,generated on Jan. 4, 2023 and filed electronically, is provided in lieuof a paper copy. This Sequence Listing is hereby incorporated byreference into the specification for its disclosures.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.AR050595, AR056394, and AR056953 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to polynucleotides encoding mini-dystrophinproteins, viral vectors comprising the same, and methods of using thesame for delivery of mini-dystrophin to a cell or a subject.

BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD) is a severe, x-linked, progressiveneuromuscular disease affecting approximately one in 3,600 to 9200 livemale births. The disorder is caused by frame shift mutations in thedystrophin gene abolishing the expression of the dystrophin protein. Dueto the lack of the dystrophin protein, skeletal muscle, and ultimatelyheart and respiratory muscles (e.g., intercostal muscles and diaphragm),degenerate causing premature death. Progressive weakness and muscleatrophy begins in childhood, starting in the lower legs and pelvisbefore spreading into the upper arms. Other symptoms include loss ofcertain reflexes, waddling gait, frequent falls, difficulty rising froma sitting or lying position, difficulty climbing stairs, changes tooverall posture, impaired breathing, and cardiomyopathy. Many childrenare unable to run rapidly or jump. The atrophied muscles, in particularthe calf muscles (and, less commonly, muscles in the buttocks,shoulders, and arms), may be enlarged by an accumulation of fat andconnective tissue, causing them to look larger and healthier than theyactually are (called pseudohypertrophy). Bone thinning and scoliosis arecommon. Ultimately, independent ambulation is lost, and a wheelchairbecomes necessary, in most cases between 12 to 15 years of age. As thedisease progresses, the muscles in the diaphragm that assist inbreathing and coughing become weaker. Affected individuals experiencebreathing difficulties, respiratory infections, and swallowing problems.Almost all DMD patients will develop cardiomyopathy. Pneumoniacompounded by cardiac involvement is the most frequent cause of death,which frequently occurs before the third decade.

Becker muscular dystrophy (BMD) has less severe symptoms than DMD, butstill leads to premature death. Compared to DMD, BMD is characterized bylater-onset skeletal muscle weakness. Whereas DMD patients arewheelchair dependent before age 13, those with BMD lose ambulation andrequire a wheelchair after age 16. BMD patients also exhibitpreservation of neck flexor muscle strength, unlike their counterpartswith DMD. Despite milder skeletal muscle involvement, heart failure fromDMD-associated dilated cardiomyopathy (DCM) is a common cause ofmorbidity and the most common cause of death in BMD, which occurs onaverage in the mid-40s.

Dystrophin is a cytoplasmic protein encoded by the dmd gene, andfunctions to link cytoskeletal actin filaments to membrane proteins.Normally, the dystrophin protein, located primarily in skeletal andcardiac muscles, with smaller amounts expressed in the brain, acts as ashock absorber during muscle fiber contraction by linking the actin ofthe contractile apparatus to the layer of connective tissue thatsurrounds each muscle fiber. In muscle, dystrophin is localized at thecytoplasmic face of the sarcolemma membrane.

First identified in 1987, the dmd gene is the largest known human geneat approximately 2.5 Mb. The gene is located on the X chromosome atposition Xp21 and contains 79 exons. The most common mutations thatcause DMD or BMD are large deletion mutations of one or more exons(60-70%), but duplication mutations (5-10%), and single nucleotidevariants, (including small deletions or insertions, single-base changes,and splice site changes accounting for approximately 25%-35% ofpathogenic variants in males with DMD and about 10%-20% of males withBMD) can also cause pathogenic dystrophin variants.

In DMD, mutations often lead to a frame shift resulting in a prematurestop codon and a truncated, non-functional or unstable protein. Nonsensepoint mutations can also result in premature termination codons with thesame result. While mutations causing DMD can affect any exon, exons 2-20and 45-55 are common hotspots for large deletion and duplicationmutations. In frame deletions result in the less severe Becker musculardystrophy (BMD), in which patients express a truncated, partiallyfunctional dystrophin.

Full-length dystrophin is a large (427 kDa) protein comprising a numberof subdomains that contribute to its function. These subdomains include,in order from the amino-terminus toward the carboxy-terminus, theN-terminal actin-binding domain, a central so-called “rod” domain, acysteine-rich domain and lastly a carboxy-terminal domain or region. Therod domain is comprised of 4 proline-rich hinge domains (abbreviated H),and 24 spectrin-like repeats (abbreviated R) in the following order: afirst hinge domain (H1), 3 spectrin-like repeats (R1, R2, R3), a secondhinge domain (H2), 16 more spectrin-like repeats (R4, R5, R6, R7, R8,R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19), a third hingedomain (H3), 5 more spectrin-like repeats (R20, R21, R22, R23, R24), andfinally a fourth hinge domain (H4). Subdomains toward thecarboxy-terminus of the protein are involved in connecting to thedystrophin-associated glycoprotein complex (DGC), a large proteincomplex that forms a critical link between the cytoskeleton and theextra-cellular matrix.

No treatment definitively halts or reverses progression of DMD.Treatment with corticosteroids is the current standard of care, but thismerely slows progression by a year or two. A number of new drugs for DMDhave recently been approved by regulators. These include ataluren, whichcauses read-through of premature stop codons, and eteplirsen, whichcauses skipping of exon 51, generating an internally deleted partiallyfunctional dystrophin. However, the mechanism of action of these drugsis not expected to help all DMD patients, and further evidence isrequired to definitively demonstrate their clinical efficacy in DMD.

With advances over the last 10-15 years in use of adeno-associated virus(AAV) mediated gene therapy to potentially treat a variety of rarediseases, there has been renewed hope and interest that AAV could beused to treat DMD and less severe dystrophinopathies (i.e., other musclediseases associated with mutations in the dmd gene). Due to limits onpayload size of AAV vectors, attention has focused on creating micro- ormini-dystrophins, smaller versions of dystrophin that eliminatenon-essential subdomains while maintaining at least some function of thefull-length protein. AAV-mediated mini-dystrophin gene therapy has shownpromise in mdx mice, an animal model for DMD, with widespread expressionin muscle and evidence of improved muscle function (See, e.g., Wang etal., J. Orthop. Res. 27:421 (2009)). When related experiments using amicro-dystrophin vector were attempted in the GRMD DMD dog model,however, powerful immunosuppressant drugs were required to achievesignificant transduction of muscle cells (Yuasa et al., Gene Ther.14:1249 (2007)). Similarly, when human DMD patients were treated withAAV vectors designed to express a mini-dystrophin, minimal protein wasdetected in only two of the six patients, whereas a T-cell responseagainst the mini-dystrophin protein was stimulated in three (Bowles, etal., Mol Ther. 20(2):443-455 (2012)).

Thus, there exists a need in the art for AAV vectors encodingmini-dystrophins that can be expressed at high levels in transducedcells of subjects with DMD while minimizing immune responses to themini-dystrophin protein.

SUMMARY OF THE INVENTION

Disclosed and exemplified herein are mini-dystrophin proteins,codon-optimized genes for expressing such mini-dystrophin proteins, AAVvectors for transducing cells with such genes, and methods of preventionand treatment using such AAV vectors, in particular for preventing andtreating dystrophinopathies in subjects in need thereof. In some ofthese embodiments, AAV vectors of the disclosure are capable of guidingproduction of significant levels of mini-dystrophin in transduced cellswhile causing no or only muted immune response against themini-dystrophin protein.

Certain non-limiting embodiments (E) of the inventions of the disclosureare set forth below. These and related embodiments are described infurther detail in the Detailed Description, including the Examples andDrawings.

E1. A mini-dystrophin protein comprising, consisting essentially of, orconsisting of the N-terminus, the Actin Binding Domain (ABD), hinge H1,rods R1 and R2, hinge H3, rods R22, R23, and R24, hinge H4, thecysteine-rich (CR) domain, and a portion of the carboxy-terminal (CT)domain of wildtype human muscle dystrophin protein (SEQ ID NO:25),wherein the CT domain does not comprise the last three amino acidresidues at the carboxy-terminus of wildtype dystrophin protein.E2. The mini-dystrophin protein of E1, wherein the N-terminus and ActinBinding Domain (ABD) together comprise, consist essentially of, orconsist of amino acid numbers 1-240 from SEQ ID NO:25; hinge H1comprises, consists essentially of, or consists of amino acid numbers253-327 from SEQ ID NO:25; rod R1 comprises, consists essentially of, orconsists of amino acid numbers 337-447 from SEQ ID NO:25; rod R2comprises, consists essentially of, or consists of amino acid numbers448-556 from SEQ ID NO:25; hinge H3 comprises, consists essentially of,or consists of amino acid numbers 2424-2470 from SEQ ID NO:25; rod R22comprises, consists essentially of, or consists of amino acid numbers2687-2802 from SEQ ID NO:25; rod R23 comprises, consists essentially of,or consists of amino acid numbers 2803-2931 from SEQ ID NO:25; rod R24comprises, consists essentially of, or consists of amino acid numbers2932-3040 from SEQ ID NO:25; hinge H4 comprises, consists essentiallyof, or consists of amino acid numbers 3041-3112 from SEQ ID NO:25; theCR domain comprises, consists essentially of, or consists of amino acidnumbers 3113-3299 from SEQ ID NO:25; and the portion of the CT domaincomprises, consists essentially of, or consists of amino acid numbers3300-3408 from SEQ ID NO:25.E3. The mini-dystrophin protein of any one of E1 and E2, wherein themini-dystrophin protein comprises, consists essentially of, or consistsof the amino acid sequence of SEQ ID NO:7.E4. A mini-dystrophin protein comprising, consisting essentially of, orconsisting of the N-terminus, the Actin Binding Domain (ABD), hinge H1,rods R1, R2, R22, R23, and R24, hinge H4, the cysteine-rich (CR) domain,and a portion of the carboxy-terminal (CT) domain of wildtype humanmuscle dystrophin protein (SEQ ID NO:25), wherein the CT domain does notcomprise the last three amino acid residues at the carboxy-terminus ofwildtype dystrophin protein.E5. The mini-dystrophin protein of E4 wherein the N-terminus and ActinBinding Domain (ABD) together comprise, consist essentially of, orconsist of amino acid numbers 1-240 from SEQ ID NO:25; hinge H1comprises, consists essentially of, or consists of amino acid numbers253-327 from SEQ ID NO:25; rod R1 comprises, consists essentially of, orconsists of amino acid numbers 337-447 from SEQ ID NO:25; rod R2comprises, consists essentially of, or consists of amino acid numbers448-556 from SEQ ID NO:25; rod R22 comprises, consists essentially of,or consists of amino acid numbers 2687-2802 from SEQ ID NO:25; rod R23comprises, consists essentially of, or consists of amino acid numbers2803-2931 from SEQ ID NO:25; rod R24 comprises, consists essentially of,or consists of amino acid numbers 2932-3040 from SEQ ID NO:25; hinge H4comprises, consists essentially of, or consists of amino acid numbers3041-3112 from SEQ ID NO:25; the CR domain comprises, consistsessentially of, or consists of amino acid numbers 3113-3299 from SEQ IDNO:25; and the portion of the CT domain comprises, consists essentiallyof, or consists of amino acid numbers 3300-3408 from SEQ ID NO:25.E6. The mini-dystrophin protein of any one of E4 and E5, wherein themini-dystrophin protein comprises, consists essentially of, or consistsof the amino acid sequence of SEQ ID NO:8.E7. A polynucleotide encoding the mini-dystrophin protein of E1-E3.E8. A polynucleotide encoding the mini-dystrophin protein of E4-E6.E9. The polynucleotide of any one of E7 and E8, wherein the nucleobasesequence thereof is assembled from the coding sequence of the nativewildtype gene encoding full-length human muscle dystrophin, an exampleof which is provided by NCBI Reference Sequence NM_004006.2.E10. The polynucleotide of E9, wherein the nucleobase sequence thereofis provided by SEQ ID NO:26.E11. The polynucleotide of any one of E7-E10, wherein the nucleobasesequence is codon-optimized.E12. The polynucleotide of E11, wherein the codon-optimization decreasesor increases the GC content compared to the wildtype sequence.E13. The polynucleotide of E11, wherein the codon-optimization decreasesor increases the number of CpG dinucleotides compared to the wildtypesequence.E14. The polynucleotide of E11, wherein the codon-optimizationeliminates one or more cryptic splice sites.E15. The polynucleotide of E11, wherein the codon-optimizationeliminates one or more ribosome entry sites other than the one at thestart of the coding sequence for the mini-dystrophin protein.E16. The polynucleotide of E11, wherein the codon-optimizationsubstitutes one or more rare codons for codons that occur with higherfrequency in the type and/or species of cell in which themini-dystrophin gene is intended to be expressed.E17. The polynucleotide of E12, wherein the codon-optimization increasesthe GC content compared to wildtype and increases the level of geneexpression by at least 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%,450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 900%, 1000%, or more.E18. The polynucleotide of E12, wherein the codon-optimization increasesthe GC content compared to wildtype at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, or more.E19. The polynucleotide of E12, wherein the GC content is about or atleast 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, ormore.E20. The polynucleotide of E13, wherein the codon-optimization decreasesor increases the number of CpG dinucleotides compared to the wildtype byabout or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, or more.E21. The polynucleotide of E20, wherein the number of CpG dinucleotides,if reduced, is reduced in an amount sufficient to fully or partiallysuppress the silencing of gene expression due to the methylation of CpGmotifs.E22. The polynucleotide of E11, wherein the codon-optimization increasesthe codon adaptation index (CAI) of the mini-dystrophin gene inreference to highly expressed human genes to a value that is at least0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81,0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93,0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.E23. The polynucleotide of any one of E11-E22, wherein the nucleobasesequence is human codon-optimized.E24. The polynucleotide of any one of E11-E22, wherein the nucleobasesequence is canine codon-optimized.E25. The polynucleotide of E23, wherein the human codon-optimizedsequence is provided by SEQ ID NO:1, or a nucleobase sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identicalthereto.E26. The polynucleotide of E23, wherein the human codon-optimizedsequence is provided by SEQ ID NO:2, or a nucleobase sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identicalthereto.E27. The polynucleotide of E24, wherein the canine codon-optimizedsequence is provided by SEQ ID NO:3, or a nucleobase sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identicalthereto.E28. A vector comprising the polynucleotide of any of any one of E7-E27.E29. The vector of E28, wherein the polynucleotide is operably linked toa genetic control region.E30. The vector of E29, wherein the genetic control region is apromoter.E31. The vector of E30, wherein the promoter is muscle-specific in beingmore active in muscle cells compared to other types of cells, such asliver cells.E32. The vector of any one of E30-E31, wherein the genetic controlregion further includes an enhancer.E33. The vector of any one of E30-E32, wherein the promoter, andenhancer if present, is from a muscle creatine kinase (CK) gene.E34. The vector of E33, wherein the CK gene is from mouse or human.E35. The vector of E33, wherein the genetic control region is the mouseCK7 enhancer and promoter.E36. The vector of any one of E29-E36, wherein the genetic controlregion comprises the nucleobase sequence selected from the group SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:16.E37. The vector of any one of E28-E36, wherein the polynucleotide isoperably linked to a transcription terminator region.E38. The vector of E37, wherein the transcription terminator regioncomprises the nucleobase sequence of SEQ ID NO:6 or SEQ ID NO:17.E39. The vector of any one of E28-E38, wherein the vector is an AAVviral vector genome and comprises flanking AAV inverted terminal repeats(ITRs).E40. The vector of E39, wherein the ITRs are both AAV2 ITRs.E41. The vector of any one of E39 and E40, wherein the nucleobasesequence of the vector is provided by a nucleobase sequence selectedfrom the group SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,and SEQ ID NO:18.E42. A recombinant AAV (rAAV) particle comprising an AAV capsid and thevector of any one of E39-E41.E43. The rAAV particle of E42, wherein the AAV capsid is the AAV9capsid.E44. A rAAV particle, comprising an AAV capsid having tropism forstriated muscle and a vector genome for expressing a humanmini-dystrophin protein.E45. The rAAV particle of E44, wherein the AAV capsid is from the AAV9serotype.E46. The rAAV particle of any one of E44 and E45, wherein the vectorgenome comprises a human codon-optimized nucleic acid sequence encodingthe human mini-dystrophin protein.E47. The rAAV particle of any one of E44-E46, wherein the humanmini-dystrophin protein comprises the following subdomains or portionsthereof from full-length human muscle dystrophin protein in order fromN-terminus to C-terminus: N-terminal domain, Actin-Binding Domain (ABD),hinge H1, rod R1, rod R2, hinge H3, rod R22, rod R23, rod R24, hinge H4,the Cysteine-Rich (CR) Domain, and a portion of the carboxy-terminal(CT) domain, wherein the portion of the CT domain does not include thelast 3 amino acids from dystrophin.E48. The rAAV particle of any one of E44-E47, wherein the humanmini-dystrophin protein comprises the amino acid sequence of SEQ IDNO:7.E49. The rAAV particle of any one of E44-E46, wherein the humanmini-dystrophin protein comprises the following subdomains or portionsthereof from full-length human muscle dystrophin protein in order fromN-terminus to C-terminus: N-terminal domain, Actin-Binding Domain (ABD),hinge H1, rod R1, rod R2, rod R22, rod R23, rod R24, hinge H4, theCysteine-Rich (CR) Domain, and a portion of the carboxy-terminal (CT)domain, wherein the portion of the CT domain does not include the last 3amino acids from dystrophin.E50. The rAAV particle of any one of E44-E46, and E49, wherein the humanmini-dystrophin protein comprises the amino acid sequence of SEQ IDNO:8.E51. The rAAV particle of any one of E44-E47, wherein the humancodon-optimized nucleic acid sequence encoding the human mini-dystrophinprotein comprises the nucleic acid sequence of SEQ ID NO:1.E52. The rAAV particle of any one of E44-E46, E49, and E50, wherein thehuman codon-optimized nucleic acid sequence encoding the humanmini-dystrophin protein comprises the nucleic acid sequence of SEQ IDNO:3.E53. The rAAV particle of any one of E44-E52, wherein the vector genomefurther comprises AAV inverted terminal repeats (ITRs) flanking thecodon-optimized nucleic acid sequence.E54. The rAAV particle of E53, wherein the AAV ITRs are AAV2 ITRs.E55. The rAAV particle of any one of E44-E54, wherein the vector genomefurther comprises a muscle-specific transcriptional regulatory elementoperably linked with the human codon optimized nucleic acid sequence.E56. The rAAV particle of E55, wherein the muscle-specifictranscriptional regulatory element is positioned between the 5′ AAV2 ITRand the human codon-optimized nucleic acid sequence.E57. The rAAV particle of any one of E55 and E56, wherein themuscle-specific transcriptional regulatory element is derived from thehuman or mouse creatine kinase (CK) gene.E58. The rAAV particle of any one of E55-E57, wherein themuscle-specific transcriptional regulatory element comprises an enhancerand a promoter.E59. The rAAV particle of any one of E55-E58, wherein themuscle-specific transcriptional regulatory element is the mouse CK7enhancer and promoter.E60. The rAAV particle of any one of E55-E59, wherein themuscle-specific transcriptional regulatory element comprises the nucleicacid sequence of SEQ ID NO:16.E61. The rAAV particle of any one of E44-E60, wherein the vector genomefurther comprises a transcription termination sequence positionedbetween the codon-optimized nucleic acid sequence and the 3′ AAV2 ITR.E62. The rAAV particle of E61, wherein the transcription terminationsequence comprises a polyadenylation signal.E63. The rAAV particle of any one of E44-E62, wherein the vector genomecomprises in 5′ to 3′ order: a first AAV2 ITR, a muscle-specifictranscriptional regulatory element operably linked to a humancodon-optimized nucleic acid sequence encoding a human mini-dystrophinprotein, a transcription termination sequence, and a second AAV2 ITR.E64. The rAAV particle of E63, wherein the muscle-specifictranscriptional regulatory element comprises the nucleic acid sequenceof SEQ ID NO:16.E65. The rAAV particle of embodiments E63 or E64, wherein the humancodon-optimized nucleic acid sequence comprises the nucleic acidsequence of SEQ ID NO:1.E66. The rAAV particle of embodiments E63-E65, wherein the transcriptiontermination sequence comprises the nucleic acid sequence of SEQ IDNO:17.E67. The rAAV particle of any one of E44-E48, E51, and E53-E66, whereinthe vector genome comprises the nucleic acid sequence of SEQ ID NO:18 orthe reverse-complement thereof.E68. The rAAV particle of any one of E44-E48, E51, and E53-E66, whereinthe vector genome consists essentially of the nucleic acid sequence ofSEQ ID NO:18 or the reverse-complement thereof.E69. The rAAV particle of any one of E44-E48, E51, and E53-E66, whereinthe vector genome consists of the nucleic acid sequence of SEQ ID NO:18or the reverse-complement thereof.E70. A recombinant AAV particle, comprising an AAV9 capsid and a vectorgenome comprising the nucleic acid sequence of SEQ ID NO:18 or thereverse complement thereof.E71. A recombinant AAV particle, comprising an AAV9 capsid and a vectorgenome consisting essentially of the nucleic acid sequence of SEQ IDNO:18 or the reverse complement thereof.E72. A recombinant AAV particle, comprising an AAV9 capsid and a vectorgenome consisting of the nucleic acid sequence of SEQ ID NO:18 or thereverse complement thereof.E73. A pharmaceutical composition comprising the rAAV particle of anyone of E42-E72 and a pharmaceutically acceptable carrier.E74. A method for treating a dystrophinopathy comprising administeringto a subject in need of treatment for a dystrophinopathy atherapeutically effective amount of the composition of E73.E75. Use of the recombinant AAV (rAAV) particle of any one of E42-E72 oruse of the composition of E73 in the preparation of a medicament fortreating a subject with a dystrophinopathy.E76. The rAAV particle of any one of E42-E72 or the composition of E73for use in the treatment of a subject having a dystrophinopathy.E77. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the dystrophinopathy is Duchenne muscular dystrophy(DMD), Becker muscular dystrophy (BMD), or DMD-associated dilatedcardiomyopathy.E78. The method, use, rAAV particle, or composition for use of any oneof E74-E77, wherein the subject is a male or female human subject.E79. The method, use, rAAV particle, or composition for use of any oneof E74-E78, wherein the subject is ambulatory when first treated with oradministered the composition.E80. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the subject is about or at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 years of age when first treated with oradministered the composition.E81. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to restore dystrophin associated protein complex at thesarcolemma of muscle cells compared to untreated controls.E82. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to improve the dystrophic histopathology in the heartcompared to untreated controls.E83. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to inhibit fibrosis in limb muscle and diaphragmcompared to untreated controls.E84. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce muscle lesion score compared to untreatedcontrols.E85. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce muscle fatigue compared to untreatedcontrols.E86. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to increase the maximum absolute or relative forelimbgrip strength of Dmd^(mdx) rats compared to untreated controls.E87. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to increase the detectable level of mini-dystrophinmRNA or protein in skeletal muscle, heart muscle or diaphragm.E88. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce average MMP-9 levels in blood of subjects towithin about 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-, 5-, 4-, 3-,or 2-fold greater than that in healthy controls.E89. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce average ALT, AST, or LDH levels in blood ofsubjects to within about 7-, 6-, 5-, 4-, 3-, or 2-fold greater than thatin healthy controls.E90. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce average total CK levels in blood of subjectsto within about 50-, 48-, 46-, 44-, 42-, 40-, 38-, 36-, 34-, 32-, 30-,28-, 26-, 24-, 22-, 20-, 18-, 16-, 14-, 12-, 10-, 9-, 8-, 7-, 6-, 5-,4-, 3-, or 2-fold greater than that in healthy controls.E91. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to increase the average 6 minute walk distance (6MWD)of subjects by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 meters compared to the average 6MWDof untreated controls 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, or 36months after administration of the vector.E92. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce the average time required to perform the 4stair climb test by at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6,1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 secondscompared to the average time of untreated controls 3, 6, 9, 12, 15, 18,21, 24, 27, 30, 33, or 36 months after administration of the vector.E93. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce the average proportion of subjects that havelost ambulation by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60% or 65% compared to the average proportion of untreatedcontrols that have lost ambulation 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, or 36 months after administration of the vector.E94. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to reduce the average fat fraction in the lowerextremities of subjects by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the average fatfraction in the lower extremities of untreated controls 3, 6, 9, 12, 15,18, 21, 24, 27, 30, 33, or 36 months after administration of the vector.E95. The method, use, rAAV particle, or composition for use of any oneof E88-E94, wherein the controls are age and sex matched to thesubjects.E96. The method, use, rAAV particle, or composition for use of any oneof E91-E94, wherein the subjects and untreated controls are stratifiedaccording to age, prior corticosteroid treatment, and/or baselineperformance on the 6MWT.E97. The method, use, rAAV particle, or composition for use of any oneof E74-E79, wherein the method, use, rAAV particle, or composition foruse is effective to cause at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of skeletalmuscle fibers of a subject to express the mini-dystrophin protein 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, or 36 months after administration ofthe vector.E98. The method, use, rAAV particle, or composition for use of any oneof E97, wherein the skeletal muscle fibers are present in a biopsyobtained from the bicep, deltoid or quadriceps muscle of the subject.E99. The method, use, rAAV particle, or composition for use of any oneof E74-E98, wherein the method, use, rAAV particle, or composition foruse causes a cellular immune response against the mini-dystrophinprotein or muscle inflammation in less than or equal to about 0%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13, 14%, 15%, 16%, 17%,18%, 19% or 20% of subjects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 or 36 months after administration of the vector.E100. The method, use, rAAV particle, or composition for use of any oneof E74-E99, wherein the method, use, rAAV particle, or composition foruse is effective without need for concomitant immune suppression intreated subjects.E101. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in areduction in serum AST, ALT, LDH, or total creatine kinase levels at 3months or 6 months post-injection compared to age matched controlsadministered only vehicle.E102. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in areduction in fibrosis in biceps femoris, diaphragm, or heart muscle at 3months or 6 months post-injection compared to age matched controlsadministered only vehicle.E103. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in anincrease in forelimb grip force at 3 months or 6 months post-injectioncompared to age matched controls administered only vehicle.E104. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in areduction in muscle fatigue as measured over 5 closely spaced trialstesting forelimb grip force at 3 months or 6 months post-injectioncompared to age matched controls administered only vehicle.E105. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in anincrease in left ventricular ejection fraction as measured usingechocardiography at 6 months post-injection compared to age matchedcontrols administered only vehicle.E106. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in anincrease in the ratio of the velocity of early to late left ventricularfilling (i.e., E/A ratio) as measured using echocardiography at 3 monthsor 6 months post-injection compared to age matched controls administeredonly vehicle.E107. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to result in adecrease in the isovolumetric relaxation time (IVRT) or the time inmilliseconds between peak E velocity and its return to baseline, whereinthe E wave deceleration time (DT) is measured using echocardiography at3 months or 6 months post-injection compared to age matched controlsadministered only vehicle.E108. The method, use, rAAV particle, or composition for use of E74-E76,wherein the subject is a Dmd^(mdx) rat and the method, use, rAAVparticle, or composition for use is effective to transduce bicepsfemoris, diaphragm, heart muscle, or other striated muscles, and expressthe mini-dystrophin protein encoded by the opti-Dys3978 gene withoutinducing a cellular immune response against the mini-dystrophin proteinby 3 months or 6 months post-injection.E109. The method, use, rAAV particle, or composition for use of any oneof E74-E76, wherein the subject is a Dmd^(mdx) rat and the method, use,rAAV particle, or composition for use is effective to partially orcompletely reverse the increase in left ventricular end-diastolicdiameter at 6 months post-injection compared to age matched controlsadministered only vehicle.E110. The method, use, rAAV particle, or composition for use of any oneof E74-E100, wherein the subject is also treated with, or thecomposition also comprises, at least a second agent effective fortreating dystrophinopathy, examples of which include an antisenseoligonucleotide that causes exon skipping of the DMD gene, ananti-myostatin antibody, an agent that promotes ribosomal read-throughof nonsense mutations, an agent that suppresses premature stop codons,an anabolic steroid, or a corticosteroid (such as, without limitation,prednisone, deflazacort, or prednisolone).E111. The method, use, rAAV particle, or composition for use of any oneof E74-E110, wherein the composition is administered systemically, suchas by intravenous injection, or locally, such as directly into a muscle.E112. The method, use, rAAV particle, or composition for use of any oneof E74-E111, wherein the dose of rAAV particles used in the method, use,rAAV particle, or composition for use is selected from the group ofdoses consisting of: 1×10¹² vg/kg, 2×10¹² vg/kg, 3×10¹² vg/kg, 4×10¹²vg/kg, 5×10¹² vg/kg, 6×10¹² vg/kg, 7×10¹² vg/kg, 8×10¹² vg/kg, 9×10¹²vg/kg, 1×10¹³ vg/kg, 2×10¹³ vg/kg, 3×10¹³ vg/kg, 4×10¹³ vg/kg, 5×10¹³vg/kg, 6×10¹³ vg/kg, 7×10¹³ vg/kg, 8×10¹³ vg/kg, 9×10¹³ vg/kg, 1×10¹⁴vg/kg, 1.5×10¹⁴ vg/kg, 2×10¹⁴ vg/kg, 2.5×10¹⁴ vg/kg, 3×10¹⁴ vg/kg,3.5×10¹⁴ vg/kg, 4×10¹⁴ vg/kg, 5×10¹⁴ vg/kg, 6×10¹⁴ vg/kg, 7×10¹⁴ vg/kg,8×10¹⁴ vg/kg, and 9×10¹⁴ vg/kg, where vg/kg stands for vector genomesper kilogram of subject body weight.E113. The composition of E73, further comprising empty capsids of thesame AAV serotype as the rAAV particle, wherein the concentration ratioof empty capsids to rAAV particles is about or at least 0.5:1, 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or more.E114. A method of expressing a mini-dystrophin protein in a cell,comprising contacting the cell with the rAAV particle of any one ofE42-E72.E115. The method of E114, wherein the cell is a muscle cell.E116. The method of E115, wherein the muscle cell is from skeletalmuscle, diaphragm, or heart.E117. A method of making the rAAV particle of any one of E42-E72,comprising introducing into a producer cell the vector of any one ofE39-E41, an AAV rep gene, an AAV cap gene, and genes for helperfunctions, incubating the cells, and purifying the rAAV particlesproduced by the cells.E118. The method of E117, wherein the producer cells are adherent.E119. The method of E117, wherein the producer cells are non-adherent.E120. The method of any one of E117-E119, wherein the vector iscontained in one plasmid, the AAV rep and cap genes are contained in asecond plasmid, and the helper function genes are contained in a thirdplasmid, where all three plasmids are introduced into the packagingcells.E121. The method of any one of E117-E120, wherein the step ofintroducing is effected by transfection.E122. The method of any one of E117-E121, wherein the producer cells areHEK 293 cells.E123. The method of any one of E117-E122, wherein the producer cells aregrown in serum free medium.E124. The method of any one of E117-E123, wherein the AAV cap geneencodes the AAV9 VP1, VP2 and VP3 proteins.E125. The method of any one of E117-E124, wherein the rAAV particles arepurified using density gradient ultracentrifugation, or columnchromatography.E126. An rAAV particle produced by the method of any one of E117-E125.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction of highly truncated mini-dystrophin genes.Wild-type muscle dystrophin has four major domains: the N-terminaldomain (N); the central rod domain, which contains 24 rod repeats (R)and four hinges (H); a cysteine-rich (CR) domain, and thecarboxy-terminal (CT) domain. The mini-dystrophin genes were constructedby deleting a large portion of the central rods and hinges and most ofthe CT domain. The mini-dystrophin genes were codon-optimized, fullysynthesized and subsequently cloned between a CMV promoter or amuscle-specific synthetic hybrid promoter at the 5′ end of the gene, anda small poly(A) sequence at the 3′ end of the gene. This gene segment,containing promoter, codon-optimized mini-dystrophin gene, and polyAsignal, was then cloned into a plasmid containing left and right AAVinverted terminal repeats (ITRs) so that the gene segment was flanked bythe ITRs.

FIG. 2 shows codon-optimization effectively enhances mini-dystrophingene expression. The top panels show immunofluorescence (IF) staining ofmini-dystrophin protein in (A) untransfected 293 cells or aftertransfection of original un-optimized (B), or optimized (C)mini-dystrophin Dys3978 vector plasmids. The bottom panels show Westernblots of the mini-dystrophin in the transfected 293 cells. Blot on theleft used an equal amount of cell lysates and shows overwhelmingexpression by the optimized cDNA. Blot on the right used a 100× dilutionof the cell lysate from 293 cells transfected with optimizedmini-dystrophin cDNA, while the non-optimized sample was not diluted.Note that the signal of the optimized one is still stronger after 100×dilution.

FIG. 3 shows IF staining of human mini-dystrophin expression indystrophin/utrophin double knockout (dKO) mice treated with AAV9 vector.Muscle and heart samples from wild-type control mice C57BL/10 (C57),untreated dKO mice, and AAV9-CMV-Hopti-Dys3978 treated dKO mice (T-dKO)were thin-sectioned and stained with an antibody that also recognizesboth the mouse wild-type dystrophin and human mini-dystrophin protein.Highly efficient expression was achieved in all samples examined.

FIG. 4 shows normalization of body weight of dKO mice as a result ofAAV9-CMV-Hopti-Dys3978 treatment. Data were obtained at 4 months of agefrom wild-type control B10 mice (C57BL/10), untreated mdx mice,untreated dKO mice, and vector-treated dKO mice.

FIG. 5 shows improvement of grip force and treadmill running of dKO miceas a result of AAV9-CMV-Hopti-Dys3978 treatment. Data were obtained at 3months of age from wild-type control B10 mice (C57BL/10), untreated mdxmice, untreated dKO mice, and vector-treated dKO mice (T-dKO).

FIGS. 6A-6B show amelioration of dystrophic pathology of dKO mice as aresult of AAV9-CMV-Hopti-Dys3978 treatment. (FIG. 6A) Cryosections (8μm) of tibialis anterior muscles from wild-type control C57BL/10 mice,untreated dKO mice, and vector-treated dKO (T-dKO) mice were subjectedto hematoxylin and eosin (H&E) staining for histopathology (10×magnification). (FIG. 6B) Quantitative analyses of muscle mass, heartmass, percentage of centrally localized nuclei and serum creatine kinaseactivities.

FIG. 7 shows survival curves of dKO mice treated with humancodon-optimized mini-dystrophin Dys3978 vector (AAV9-CMV-Hopti-Dys3978)compared to untreated dKO mice and wildtype mice. Greater than 50% ofthe treated dKO mice survived longer than 80 weeks (duration of theexperiment).

FIG. 8 shows improvement in cardiac functions of dKO mice as a result ofAAV9-CMV-Hopti-Dys3978 treatment. Hemodynamic analysis was performed onwild-type control C57BL/10 mice, untreated mdx mice, and AAV9vector-treated dKO mice. The untreated dKO mice were too sick to sustainthe procedure. Data were collected from the three groups of mice withoutor with dobutamine challenge.

FIGS. 9A-9B show improvement in electrocardiography (ECG) of dKO mice asa result of AAV9-CMV-Hopti-Dys3978 treatment. (FIG. 9A) The PR intervalof the ECG was improved in vector-treated dKO mice. (FIG. 9B)Quantitative data of the analysis. The experiment was done to carefullymonitor the heart rate of the three groups so that the ECG was notaffected by the variation in heart rate. *p<0.05.

FIG. 10 shows a comparison of the non-tissue specific CMV promoter andthe muscle-specific hCK promoter in driving human codon-optimizedmini-dystrophin Dys3978 in mdx mice after tail vein injection ofAAV9-Hopti-Dys3978 vectors containing CMV or hCK promoter. Using IFstaining, the human mini-dystrophin Dys3978 showed robust expression inlimb muscle and heart muscle as well. It appeared that the hCK promoterwas more effective over the CMV promoter.

FIG. 11 shows magnetic resonance imaging (MRI) images of the hind limbof GRMD dog “Jelly” after isolated limb vein perfusion of theAAV9-CMV-Hopti-Dys3978 vector. The vector was infused with pressure inthe right hind leg which had a tight tourniquet placed at the groinarea. The whitish signals indicated vector solution retention in theperfused limb.

FIG. 12 shows IF staining of human mini-dystrophin Dys3978 expression at2 months post vector injection in GRMD dog “Jelly.” Biopsy samples of 5different muscle groups in both right and left hind legs were examined.The non-injected left leg also had detectable dys3978, suggesting thatthe AAV9 vector had traveled from the site of injection to thecontralateral leg.

FIG. 13 shows IF staining of human mini-dystrophin Dys3978 expression at7 months post vector injection in GRMD dog “Jelly.” Biopsy samples of 4different muscle groups in both right and left hind legs were examined.The non-injected left leg also had detectable Dys3978, suggesting thatthe AAV9 vector had traveled from the site of injection to thecontralateral leg. Western blot analysis of Dys3978 was done on the samesamples.

FIG. 14 shows IF staining of human mini-dystrophin Dys3978 expression at12 months post vector injection in GRMD dog “Jelly.” Biopsy samples of 4different muscle groups in both right and left hind legs and 1 sample inthe forelimb were examined. The non-injected left leg also haddetectable Dys3978, suggesting that the AAV9 vector had traveled fromthe site of injection to the contralateral leg.

FIG. 15 shows IF staining of human mini-dystrophin Dys3978 expression at2 years post vector injection in GRMD dog “Jelly.” Biopsy samples of 2different muscle groups in both right and left hind legs were examined.Note the non-injected left leg appeared to have more detectable Dys3978than the injected leg.

FIG. 16 shows IF staining of human mini-dystrophin Dys3978. Biopsysamples of two additional (compared with FIG. 15 ) muscle groups in bothright and left hind legs and one sample in the forelimb were examinedfrom GRMD dog “Jelly.” Samples were also collected at 2 years postvector injection.

FIG. 17 shows IF staining of human mini-dystrophin Dys3978 at 4 yearspost vector injection in the non-injected left hind leg from GRMD dog“Jelly.”

FIG. 18 shows IF staining of human mini-dystrophin Dys3978 at greaterthan 8 years post vector injection in GRMD dog “Jelly.” Necropsy musclesamples of 5 different muscle groups and heart were examined.

FIG. 19 shows IF staining of human mini-dystrophin Dys3978 andendogenous revertant dystrophin at greater than 8 years post vectorinjection in GRMD dog “Jelly.” Necropsy muscle samples of threedifferent muscle groups were stained with an antibody that recognizedboth human and dog dystrophin (upper panel) or an antibody that onlyrecognized dog revertant dystrophin (lower panel). The revertantdystrophin positive myofibers were highlighted by arrows. Revertantfibers are rare muscle fibers that stain positively for dystrophinprotein that occur in human DMD patients, as well as the mdx mouse andGRMD dogs. The precise mechanism by which revertant fibers occur is notcompletely understood, but may involve exon skipping in rare musclecells that produces a shortened dystrophin with the epitopes recognizedby antibody probes. See, for example, Lu, Q L, et al., J Cell Biol148:985-96 (2000).

FIG. 20 shows Western blot analyses of human mini-dystrophin Dys3978present in muscle samples of GRMD dog “Jelly” at necropsy more than 8years after AAV9 vector injection. Western blot showed humanmini-dystrophin Dys3978 was present in all skeletal muscles examined.Muscle from an age and sex matched normal dog named “Molly” was used asa positive control with serial 2-fold dilutions to indicate thequantitation of dystrophin protein. The molecular weight of wildtypefull length dystrophin is about 400 kDa while the mini-dystrophinDys3978 protein is about 150 kDa.

FIG. 21 shows muscle contractile force improvement in GRMD dog “Jelly”after injection of the AAV9-CMV-Hopti-Dys3978 vector and body wide geneexpression. The top curve represents the muscle force of a normal dog,while the bottom curve represents the muscle force of the untreated GRMDdog. The two curves extended into more time points represents the muscleforce of dog “Jelly.” Two more GRMD dogs treated withAAV9-CMV-canine-mini-dystrophin Dys3849 vector (Wang, et al., PNAS97(25):13714-9 (2000)) were also examined for muscle force, and showedimprovement (“Jasper” and “Peridot”).

FIG. 22 shows muscle biopsy IF staining of human mini-dystrophinexpression at 4 months post AAV9-hCK-Copti-Dys3978 vector injection inGRMD dog “Dunkin.” The vector was delivered by intravenous injection toachieve body wide gene expression. Biopsy samples of 4 different musclegroups in the hind limbs were examined. Note nearly uniformmini-dystrophin Dys3978 detected in all muscle groups.

FIG. 23 shows IF staining of human mini-dystrophin expression at 14months post AAV9-hCK-Copti-Dys3978 vector injection in GRMD dog“Dunkin.” Necropsy samples were taken and examined. Note widespread androbust levels of mini-dystrophin Dys3978 detected in heart and allmuscle groups. Magnification 4×.

FIG. 24 shows IF staining of diaphragm muscle with robust levels ofhuman mini-dystrophin detected at 14 months post AAV9-hCK-Copti-Dys3978vector injection in GRMD dog “Dunkin.”

FIG. 25 shows IF staining of peroneus longus muscle with robust levelsof human mini-dystrophin detected at 14 months postAAV9-hCK-Copti-Dys3978 vector injection in GRMD dog “Dunkin.”

FIG. 26 shows IF staining of semi-membranosus muscle with robust levelsof human mini-dystrophin detected at 14 months postAAV9-hCK-Copti-Dys3978 vector injection in GRMD dog “Dunkin.”

FIG. 27 shows IF staining of heart left ventricle (LV) muscle withrobust levels of human mini-dystrophin detected at 14 months postAAV9-hCK-Copti-Dys3978 vector injection in GRMD dog “Dunkin.”

FIG. 28 shows detection by Western blot of human mini-dystrophin Dys3978in muscle samples of GRMD dog “Dunkin” at 4 months and 14 months postvector injection. Muscle from an age matched normal dog was used as apositive control with serial 2-fold dilutions to indicate thequantitation of dystrophin protein. The molecular weight of wildtypefull length dystrophin is about 400 kDa while the mini-dystrophinDys3978 is about 150 kDa. Note that no mini-dystrophin Dys3978 wasdetected in the liver.

FIG. 29 shows detection by Western blot of human mini-dystrophin Dys3978expression in heart (LV) sample of GRMD dog “Dunkin” at 14 months postvector injection. Heart sample from an age-matched normal dog was usedas a positive control with serial 2-fold dilutions to indicate thequantitation of dystrophin protein.

FIG. 30 shows restoration of dystrophin associated protein complex asshown by IF staining of human mini-dystrophin Dys 3978 as well asgamma-sarcoglycan (r-SG) of various muscle groups.

FIG. 31 shows analysis of AAV9-CMV-Copti-Dys3978 vector DNA copy invarious muscle and tissues. Quantitative PCR (qPCR) was performed todetermine the AAV vector DNA genome copy numbers, which were normalizedon a per diploid cell basis.

FIG. 32 shows improvement of dystrophic histopathology in the heart ofAAV9-CMV-Copti-Dys3978 vector GRMD dog “Dunkin” compared to age-matchednormal and untreated GRMD dog. HE staining.

FIG. 33 shows improvement of dystrophic histopathology in the diaphragmmuscle of AAV9-CMV-Copti-Dys3978 vector GRMD dog “Dunkin.” Compared toage-matched normal and untreated GRMD dog. HE staining.

FIG. 34 shows improvement of dystrophic histopathology in the limbmuscles of AAV9-CMV-Copti-Dys3978 vector GRMD dog “Dunkin” compared toage-matched untreated GRMD dog. HE staining.

FIG. 35 shows inhibition of fibrosis in limb muscle and diaphragm ofGRMD dog “Dunkin” compared to age-matched untreated GRMD dog. MasonTrichrome blue staining.

FIG. 36A provides photomicrographs showing immunolabeling withanti-dystrophin DYSB antibody of biceps femoris muscle obtained from aWT rat mock treated with PBS (left panel), a mock treated DMD rat(central panel), and a Dmd^(mdx) rat treated withAAV9.hCK.Hopti-Dys3978.spA vector (right panel). The dark outline aroundthe fibers shows the subsarcolemmal localization of the dystrophin in WTrat and mini-dystrophin in vector treated Dmd^(mdx) rat.

FIG. 36B provides photomicrographs showing haematoxylin and eosin (HES)stained biceps femoris muscle obtained from a mock treated WT rat (leftpanel), a mock treated Dmd^(mdx) rat (central panel) and a DMD rattreated with AAV9.hCK.Hopti-Dys3978.spA vector (right panel). Cluster ofnecrotic fibers (*) and endomysial mild fibrosis (black arrowhead) areshown.

FIG. 36C provides photomicrographs showing immunolabeling withanti-dystrophin DYSB antibody of cardiac muscle obtained from a mocktreated WT rat (left panel), a mock treated Dmd^(mdx) rat (centralpanel) and a Dmd^(mdx) rat treated with AAV9.hCK.Hopti-Dys3978.spAvector (right panel). The dark outline around the fibers shows thesubsarcolemmal localization of the dystrophin in WT rat andmini-dystrophin in vector treated Dmd^(mdx) rat.

FIG. 36D provides photomicrographs showing HES stained cardiac muscleobtained from a mock treated WT rat (left panel), a mock treatedDmd^(mdx) rat (central panel) and a Dmd^(mdx) rat treated withAAV9.hCK.Hopti-Dys3978.spA vector (right panel). A focus of fibrosis(open arrowhead) is shown in the center panel, and a focus ofmononuclear cell infiltration is illustrated in the right panel.

FIG. 37 shows average body weight in grams of WT rats treated withvehicle (buffer) and Dmd^(mdx) rats treated with vehicle and increasingdoses of AAV9.hCK.Hopti-Dys3978.spA vector over time to 25 weeks afterdosing. “WT” refers to wild type rats; “DMD” refers to Dmd^(mdx) rats;“n” refers to sample size; “D” refers to number days since dosing; “W”refers to number of weeks since dosing; “E” is notation for thespecified coefficient times ten raised to the power of the specifiedexponent (thus, “1E13” stands for 1×10¹³, “3E13” stands for 3×10¹³,“1E14” stands for 1×10¹⁴, and “3E14” stands for 3×10¹⁴); “vg/kg” standsfor vector genomes per kilogram body weight; and “w/o HAS” refers to atreatment arm where the vector was administered in PBS without humanserum albumin. On the right side of the graph, at 25 weeks, the order ofaverage body weight data from top to bottom is the same as the top tobottom order of the treatment arms listed in the legend (except fortreatment of Dmd^(mdx) rats with 1×10¹⁴ vg/kg vector administered invehicle without HSA, for which data collection ended at 13 weeks fromthe study start). These same abbreviations are used in other figuresherein.

FIG. 38A provides exemplary photomicrographs of skeletal muscle fromDmd^(mdx) rats stained for histological examination illustrating asemi-quantitative scoring scheme used to estimate the degree of severityof muscle lesions caused by the absence of dystrophin. In skeletalmuscle, such as that illustrated, a score of 0 corresponded to theabsence of lesions; 1 corresponded to the presence of some regenerativeactivity as evidenced by centronucleated fibers and small foci ofregeneration; 2 corresponded to the presence of degenerated fibers,isolated or in small clusters; and 3 corresponded to tissue remodelingand fiber replacement by fibrotic or adipose tissue. Scoring for heartused different criteria as explained in the text.

FIG. 38B shows total DMD lesion scores for rats (that is, average oflesion subscores for biceps femoris, pectoralis, diaphragm and cardiacmuscles) at 3 months post-injection are shown, individually as well asthe mean among all rats in each treatment arm, and compared to show avector dose-responsive reduction in lesion score. “WT mock” refers to WTrats treated with vehicle, “KO mock” refers to Dmd^(mdx) rats treatedwith vehicle, “KO 1E13”, “3E13”, and “1E14”, refer to Dmd^(mdx) ratstreated with the indicated doses of AAV9.hCK.Hopti-Dys3978.spA vector invg/kg. Letters above bars indicate that the underlying data is notstatistically different from other bars over which the same lettersappear. Conversely, bars over which different letters appear arestatistically different from each other. Statistics were calculatedusing the Kruskal-Wallis and Dunn's tests.

FIG. 39A provides representative sections from biceps femoris musclesamples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls. Samples weredual labeled with an antibody that specifically binds to full length ratdystrophin and human mini-dystrophin, and wheat germ agglutininconjugate which stains connective tissue. Top panel are micrographs fromanimals sacrificed at 3 months post-injection. Bottom panel aremicrographs from animals sacrificed at 6 months post-injection.

FIG. 39B provides percent fibers in random sections from biceps femorismuscle samples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, that stainedpositive for presence of dystrophin protein. Data for 3 and 6 monthspost-injection are included. Letters above bars indicate that theunderlying data is not statistically different from other bars overwhich the same letters appear. Conversely, bars over which differentletters appear are statistically different from each other. Statisticswere calculated using ANOVA analysis and Fisher's post-hoc bilateraltest.

FIG. 39C provides percent area in random sections of biceps femorismuscle samples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, that stainedpositive for presence of connective tissue. Data for 3 and 6 monthspost-injection are included. Letters above bars indicate that theunderlying data is not statistically different from other bars overwhich the same letters appear. Conversely, bars over which differentletters appear are statistically different from each other. Statisticswere calculated using ANOVA analysis and Fisher's post-hoc bilateraltest.

FIG. 40A provides representative sections from diaphragm muscle samplesfrom Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, sacrificed at3 months post-injection. Samples were dual labeled with an antibody thatspecifically binds to full length rat dystrophin and humanmini-dystrophin, and wheat germ agglutinin conjugate which stainsconnective tissue.

FIG. 40B provides percent fibers in random sections from diaphragmmuscle samples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, that stainedpositive for presence of dystrophin. Data for 3 and 6 monthspost-injection are included. Letters above bars indicate that theunderlying data is not statistically different from other bars overwhich the same letters appear. Conversely, bars over which differentletters appear are statistically different from each other. Statisticswere calculated using ANOVA analysis and Fisher's post-hoc bilateraltest.

FIG. 40C provides percent area in random sections of diaphragm musclesamples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, that stainedpositive for presence of connective tissue. Data for 3 and 6 monthspost-injection are included. Letters above bars indicate that theunderlying data is not statistically different from other bars overwhich the same letters appear. Conversely, bars over which differentletters appear are statistically different from each other. Statisticswere calculated using ANOVA analysis and Fisher's post-hoc bilateraltest.

FIG. 41A shows representative transverse sections of heart at one-thirdfrom the apex taken from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector (top panel), and negative controls(bottom panel), sacrificed at 3 months and 6 months post-injection.Histology sections were stained with picrosirius red to permitvisualization of connective tissue. The middle panel containsrepresentative sections of heart muscle taken from vector and vehicletreated Dmd^(mdx) rats dual labeled with an antibody that specificallybinds to full length rat dystrophin and human mini-dystrophin, and wheatgerm agglutinin conjugate which stains connective tissue.

FIG. 41B provides percent fibers in random sections from heart musclesamples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, stained forpresence of dystrophin protein. Data for 3 and 6 months post-injectionare included. Letters above bars indicate that the underlying data isnot statistically different from other bars over which the same lettersappear. Conversely, bars over which different letters appear arestatistically different from each other. Statistics were calculatedusing ANOVA analysis and Fisher's post-hoc bilateral test.

FIG. 41C provides percent area in random sections of heart musclesamples from Dmd^(mdx) rats treated with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector, and negative controls, stained forpresence of connective tissue. Data for 3 and 6 months post-injectionare included. Letters above bars indicate that the underlying data isnot statistically different from other bars over which the same lettersappear. Conversely, bars over which different letters appear arestatistically different from each other. Statistics were calculatedusing ANOVA analysis and Fisher's post-hoc bilateral test.

FIG. 42A provides data regarding muscle fatigue in Dmd^(mdx) ratstreated with increasing doses of AAV9.hCK.Hopti-Dys3978.spA vectorcompared to Dmd^(mdx) and WT rats treated with vehicle measured byrepeating five closely spaced grip strength tests. Tests were conducted3 months post-injection in rats injected at 7-9 weeks of age, or whenthe rats were approximately 4.5 months old. Graph shows the decrease inforelimb grip force measured between trials 1 and 5 (expressed aspercentage of trial 1 force). Results are represented as mean±SEM.Statistics compare Dmd^(mdx) rats treated with vector against WT ratsreceiving vehicle (*p<0.05; ***p<0.001), and Dmd^(mdx) rats receivingvehicle (

<0.01;

p<0.001), both as negative controls.

FIG. 42B provides data regarding muscle fatigue in Dmd^(mdx) ratstreated with increasing doses of AAV9.hCK.Hopti-Dys3978.spA vectorcompared to Dmd^(mdx) and WT rats treated with vehicle measured byrepeating five closely spaced grip strength tests. Tests were conducted6 months post-injection in rats injected at 7-9 weeks of age, or whenthe rats were approximately 7.5 months old. Graph shows the decrease inforelimb grip force measured between trials 1 and 5 (expressed aspercentage of trial 1 force). Results are represented as mean±SEM.

FIG. 43 provides left ventricular (LV) end-diastolic diameter measuredduring diastole from long-axis images obtained by M-modeechocardiography 6 months post-injection in WT and Dmd^(mdx) ratsadministered vehicle or AAV9.hCK.Hopti-Dys3978.spA vector. Descriptivestatistics shown are mean±SEM.

FIG. 44 provides ejection fractions measured during diastole fromlong-axis images obtained by M-mode echocardiography 6 monthspost-injection in WT and Dmd^(mdx) rats administered vehicle orAAV9.hCK.Hopti-Dys3978.spA vector. Descriptive statistics shown aremean±SEM, and the “$” symbol indicates a statistically significantdifference between the data over which it is placed and the data forDmd^(mdx) rats treated with vehicle (buffer) (p<0.05).

FIG. 45A provides E/A ratios measured using pulsed Doppler with anapical four-chamber orientation 3 months post-injection in WT andDmd^(mdx) rats administered vehicle or AAV9.hCK.Hopti-Dys3978.spAvector. Descriptive statistics shown are mean±SEM, and the “*” symbolindicates a statistically significant difference between the data overwhich it is placed and the data for WT rats treated with vehicle(buffer) (p<0.05).

FIG. 45B provides E/A ratios measured using pulsed Doppler with anapical four-chamber orientation 6 months post-injection in WT andDmd^(mdx) rats administered vehicle or AAV9.hCK.Hopti-Dys3978.spAvector. Descriptive statistics shown are mean SEM, and the “**” symbolindicates a statistically significant difference between the data overwhich it is placed and the data for WT rats treated with vehicle(buffer) (p<0.01).

FIG. 46A provides isovolumetric relaxation time measured using pulsedDoppler with an apical four-chamber orientation 3 months post-injectionin WT and Dmd^(mdx) rats administered vehicle orAAV9.hCK.Hopti-Dys3978.spA vector. Descriptive statistics shown aremean±SEM.

FIG. 46B provides isovolumetric relaxation time measured using pulsedDoppler with an apical four-chamber orientation 6 months post-injectionin WT and Dmd^(mdx) rats administered vehicle orAAV9.hCK.Hopti-Dys3978.spA vector. Descriptive statistics shown aremean±SEM, and the “$” symbol indicates a statistically significantdifference between the data over which it is placed and the data forDmd^(mdx) rats treated with vehicle (buffer) (p<0.05).

FIG. 47 provides deceleration time measured using pulsed Doppler with anapical four-chamber orientation 6 months post-injection in WT andDmd^(mdx) rats administered vehicle or AAV9.hCK.Hopti-Dys3978.spAvector. Descriptive statistics shown are mean±SEM, and the “*” symbolindicates a statistically significant difference between the data overwhich it is placed and the data for WT rats treated with vehicle(buffer) (p<0.05).

FIG. 48A shows effect in Dmd^(mdx) rats of increasing doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood AST levels 3 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)as a negative control (**p<0.01, *p<0.05).

FIG. 48B shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood AST levels 6 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)as a negative control (***p<0.001, **p<0.01).

FIG. 49A shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood ALT levels 3 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)(***p<0.001, *p<0.05), or against Dmd^(mdx) rats that received buffer(##p<0.01, #p<0.05), as negative controls.

FIG. 49B shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood ALT levels 6 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)as a negative control (**p<0.01).

FIG. 50A shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood LDH levels 3 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)(***p<0.001, **p<0.01), or against Dmd^(mdx) rats that received buffer(#p<0.05), as negative controls.

FIG. 50B shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood LDH levels 6 monthspost-injection. Results are represented as mean±SEM. Statisticalanalyses were performed using the non-parametric Kruskal Wallis test anda post-hoc Dunn's multiple comparison test. Statistics compare Dmd^(mdx)rats treated with vector against WT rats that received buffer (vehicle)as a negative control (**p<0.01).

FIG. 51A shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood total creatine kinase (CK)levels 3 months post-injection. Results are represented as mean±SEM.Statistical analyses were performed using the non-parametric KruskalWallis test and a post-hoc Dunn's multiple comparison test. Statisticscompare Dmd^(mdx) rats treated with vector against WT rats that receivedbuffer (vehicle) (**p<0.01), or compare Dmd^(mdx) rats dosed with 3×10¹⁴vg/kg vector against Dmd^(mdx) rats that received buffer or 1×10¹³ vg/kgvector (##p<0.01).

FIG. 51B shows effect in Dmd^(mdx) rats of different doses ofAAV9.hCK.Hopti-Dys3978.spA vector on blood total creatine kinase (CK)levels 6 months post-injection. Results are represented as mean±SEM.Statistical analyses were performed using the non-parametric KruskalWallis test and a post-hoc Dunn's multiple comparison test. Statisticscompare Dmd^(mdx) rats treated with vector against WT rats that receivedbuffer (vehicle) as a negative control (***p<0.001, **p<0.01, *p<0.05),or compare Dmd^(mdx) rats dosed with 3×10¹⁴ vg/kg vector againstDmd^(mdx) rats that received 1×10¹³ vg/kg vector ($p<0.05).

FIG. 52A provides total creatine kinase (CK) evolution between day ofinjection (D0) of vehicle of vector and sacrifice 3 monthspost-injection. Solid bars indicate data from D0, whereas hatched barsindicate data at 3 months. Results are represented as mean±SEM.

FIG. 52B provides total creatine kinase (CK) evolution between day ofinjection (D0) of vehicle of vector and sacrifice 6 monthspost-injection. Solid bars indicate data from D0, whereas hatched barsindicate data at 6 months. Results are represented as mean±SEM.

FIG. 53A provides average absolute maximum forelimb grip strength ofolder Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Tests were conducted 3 months post-injection inrats injected at 4 months of age, or when the rats were approximately 7months old. Results are represented as mean±SEM. Statistics compareDmd^(mdx) rats treated with vector against Dmd^(mdx) rats treated withvehicle (*p<0.01).

FIG. 53B provides average maximum forelimb grip strength relative tobody weight of older Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Tests were conducted 3 months post-injection inrats injected at 4 months of age, or when the rats were approximately 7months old. Results are represented as mean±SEM. Statistics compareDmd^(mdx) rats treated with vector against Dmd^(mdx) rats treated withvehicle (*p<0.01).

FIG. 53C shows evolution of forelimb grip force as a measure of musclefatigue in older Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Test was conducted by measuring average maximumgrip force 5 times with short intervals between each trial. Tests wereconducted 3 months post-injection in rats injected at 4 months of age,or when the rats were approximately 7 months old. Results are providedrelative to body weight and as the mean±SEM. Statistics compareDmd^(mdx) rats treated with vector against WT rats receiving vehicle(*p<0.05) and Dmd^(mdx) rats receiving vehicle (

p<0.01), and compare later trials against trial 1 in vehicle treatedDmd^(mdx) rats (§§p<0.01, §§§p<0.001).

FIG. 54A provides average absolute maximum forelimb grip strength ofolder Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Tests were conducted 3 months post-injection inrats injected at 6 months of age, or when the rats were approximately 9months old. Results are represented as mean±SEM. Statistics compareDmd^(mdx) rats treated with vehicle against WT rats treated with vehicle(**p<0.01).

FIG. 54B provides average maximum forelimb grip strength relative tobody weight of older Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Tests were conducted 3 months post-injection inrats injected at 6 months of age, or when the rats were approximately 9months old. Results are represented as mean±SEM. Statistics compareDmd^(mdx) rats treated with vehicle against WT rats treated with vehicle(*p<0.05) or Dmd^(mdx) rats treated with vector against Dmd^(mdx) ratstreated with vehicle (

p<0.05).

FIG. 54C shows evolution of forelimb grip force as a measure of musclefatigue in older Dmd^(mdx) rats treated with 1×10¹⁴ vg/kgAAV9.hCK.Hopti-Dys3978.spA vector compared to Dmd^(mdx) and WT ratstreated with vehicle. Test was conducted by measuring average maximumgrip force 5 times with short intervals between each trial. Tests wereconducted 3 months post-injection in rats injected at 6 months of age,or when the rats were approximately 9 months old. Results are providedrelative to body weight and as the mean±SEM. Statistics compareDmd^(mdx) rats treated with vector against Dmd^(mdx) rats receivingvehicle (

p<0.05), Dmd^(mdx) rats treated with vehicle against WT rats receivingvehicle (**p<0.01, ***p<0.001), and trial 5 against trial 1 in vehicletreated Dmd^(mdx) rats (§§p<0.01).

FIGS. 55A-55C provide an alignment between the amino acid sequences ofthe mini-dystrophin protein Δ3990 (SEQ ID NO:27) and the mini-dystrophinprotein Dys3978 (SEQ ID NO:7).

FIGS. 56A-56I provide an alignment between the nucleic acid sequenceencoding mini-dystrophin Δ3990 (SEQ ID NO:28), which is derived from thewildtype nucleic acid sequence encoding human dystrophin protein, andthe human codon-optimized nucleic acid sequence encoding mini-dystrophinDys3978 (called Hopti-Dys3978; SEQ ID NO:1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. This invention may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinvention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 CFR § 1.822 and established usage. See,e.g., PatentIn User Manual, 99-102 (November 1990) (U.S. Patent andTrademark Office).

Except as otherwise indicated, standard methods known to those skilledin the art may be used for the construction of recombinant parvovirusand AAV (rAAV) constructs, packaging vectors expressing the parvovirusRep and/or Cap sequences, and transiently and stably transfectedpackaging cells. Such techniques are known to those skilled in the art.See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2ndEd. (Cold Spring Harbor, N Y, 1989); AUSUBEL et al., CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley &Sons, Inc., New York).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

To illustrate further, if, for example, the specification indicates thata particular amino acid can be selected from A, G, I, L and/or V, thislanguage also indicates that the amino acid can be selected from anysubset of these amino acid(s) for example A, G, I or L; A, G, I or V; Aor G; only L; etc. as if each such subcombination is expressly set forthherein. Moreover, such language also indicates that one or more of thespecified amino acids can be disclaimed. For example, in particularembodiments the amino acid is not A, G or I; is not A; is not G or V;etc. as if each such possible disclaimer is expressly set forth herein.

Definitions

The following terms are used in the description herein and the appendedclaims.

The singular forms “a” and “an” are intended to include the plural formsas well, unless the context clearly indicates otherwise.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of the length of a polynucleotide orpolypeptide sequence, dose, time, temperature, and the like, is meant toencompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “adeno-associated virus” (AAV), includes but isnot limited to, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3,including types 3A and 3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAVtype 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9),AAV type 10 (AAV10), AAV type 11 (AAV 11), AAV type 12 (AAV12), AAV type13 (AAV13), Avian AAV ATCC VR-865, Avian AAV strain DA-1, Bb1, Bb2, Ch5,Cy2, Cy3, Cy4, Cy5, Cy6, Hu1, Hu10, Hu11, Hu13, Hu15, Hu16, Hu17, Hu18,Hu19, Hu2, Hu20, Hu21, Hu22, Hu23, Hu24, Hu25, Hu26, Hu27, Hu28, Hu29,Hu3, Hu31, Hu32, Hu34, Hu35, Hu37, Hu39, Hu4, Hu40, Hu41, Hu42, Hu43,Hu44, Hu45, Hu46, Hu47, Hu48, Hu49, Hu51, Hu52, Hu53, Hu54, Hu55, Hu56,Hu57, Hu58, Hu6, Hu60, Hu61, Hu63, Hu64, Hu66, Hu67, Hu7, Hu9, HuLG15,HuS17, HuT17, HuT32, HuT40, HuT41, HuT70, HuT71, HuT88, Pi1, Pi2, Pi3,Rh1, Rh10, Rh13, Rh2, Rh25, Rh32, Rh33, Rh34, Rh35, Rh36, Rh37, Rh38,Rh40, Rh43, Rh48, Rh49, Rh50, Rh51, Rh52, Rh53, Rh54, Rh55, Rh57, Rh58,Rh61, Rh62, Rh64, Rh74, Rh8, snake AAV, avian AAV, bovine AAV, canineAAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, AAV1.1, AAV2.5,AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015/013313),AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, and any other AAV nowknown or later discovered. see, e.g., Fields et al., VIROLOGY, volume 2,chapter 69 (4th ed., Lippincott-Raven Publishers). Capsids may bederived from a number of AAV serotypes disclosed in U.S. Pat. No.7,906,111; Gao et al., 2004, J. Virol. 78:6381; Moris et al., 2004,Virol. 33:375; WO 2013/063379; WO 2014/194132; and include true type AAV(AAV-TT) variants disclosed in WO 2015/121501, and RHM4-1, RHM15-1through RHM15-6, and variants thereof, disclosed in WO 2015/013313, andone skilled in the art would know there are likely other variants notyet identified that perform the same or similar function, or may includecomponents from two or more AAV capsids. A full complement of AAV capproteins includes VP1, VP2, and VP3. The open reading frame comprisingnucleotide sequences encoding AAV capsid proteins may comprise less thana full complement AAV cap proteins or the full complement of AAV capproteins may be provided.

and any other AAV now known or later discovered. See, e.g., FIELDS etal., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-RavenPublishers). A number of relatively new AAV serotypes and clades havebeen identified (See, e.g., Gao et al., (2004) J. Virol. 78:6381; Moriset al., (2004) Virol. 33-:375).

AAV is a small non-enveloped virus with an icosahedral capsid about20-30 nm in diameter. AAV are not able to replicate without thecontribution of so-called helper proteins from other viruses (e.g.,adenovirus, herpes simplex virus, vaccinia virus and humanpapillomavirus), and so were placed into a special genus, calleddependovirus (because they depend on other viruses for replication)within the family of parvoviridae. Although many different serotypes ofAAV have been discovered, and many humans produce antibodies against oneor more AAV serotypes (suggesting widespread history of AAV infection),no diseases are known to be caused by AAV suggesting AAV isnon-pathogenic in humans.

Although many different AAV serotypes have been discovered, one of thebest characterized is AAV2, and the following discussion of AAV biologyfocuses on some of what has been learned regarding AAV2. The life cycleof other AAV serotypes is believed to be similar, although the detailsmay differ. The particular details by which AAV2 or any other AAVserotype infect and replicate inside cells are provided merely to aid inthe understanding of the inventions disclosed herein, and are notintended to limit their scope in any way. Even if some of thisinformation is later found to be incorrect or incomplete, it should notbe construed as detracting from the utility or enablement of theinventions disclosed and claimed herein. Further information about AAVlifecycle can be found in M. Goncalves, Virol J 2:43 (2005), MD Weitzmanand RM Linden, Adeno-Associated Virus Biology, Ch. 1, pp. 1-23,Adeno-Associated Virus Methods and Protocols, Ed. RO Snyder and PMoullier, Humana Press (2011), GE Berry and A Asokan, Curr Opin Virol21:54-60 (2016), and references cited therein.

The wild type genome of AAV2 is linear DNA approximately 4.7 kilobasesin length. Although mostly single-stranded, the 5′ and 3′ ends of thegenome consist of so-called inverted terminal repeats (ITR), each 145basepairs long and containing palindromic sequences that self-annealthrough classic Watson-Crick base-pairing to form T-shaped hairpinstructures. One of these structures contains a free 3′ hydroxyl groupthat, relying on cellular DNA polymerases, permits initiation of viralDNA replication through a self-priming strand-displacement mechanism.See, for example, M. Goncalves, Adeno-associated virus: from defectivevirus to effective vector, Virology J 2:43 (2005). Due to the mechanismby which the single-stranded viral genomes are replicated and thenpackaged into capsids in infected cells, plus (sense or coding) andminus (antisense or non-coding) strands are packaged with equalefficiency into separate particles.

In addition to the flanking ITRs, the wild type AAV2 genome contains twogenes, rep and cap, that code respectively for four replication proteins(Rep 78, Rep 68, Rep 52, and Rep 40) and three capsid proteins (VP1,VP2, and VP3) through efficient use of alternative promoters andsplicing. The large replication proteins, Rep 78 and 68, aremultifunctional and play a role in AAV transcription, viral DNAreplication, and site-specific integration of the viral genome intohuman chromosome 19. The smaller Rep proteins have been implicated inpacking the viral genome into the viral capsids in infected cell nuclei.The three capsid proteins are produced through a combination ofalternative splicing and use of alternative translational start sites,so that all three proteins share sequence towards their carboxy-termini,but VP2 includes additional amino-terminal sequence absent from VP3, andVP1 includes additional amino-terminal sequence absent from both VP2 andVP3. It is estimated that capsids contain a total of 60 capsid proteinsin an approximate VP1:VP2:VP3 stoichiometry of 1:1:10, although theseratios can apparently vary.

Despite its relatively small size, and therefore capacity to carryheterologous genes, AAV has been identified as a leading viral vectorfor gene therapy. Advantages of using AAV compared to other viruses thathave been proposed as gene therapy vectors include the ability of AAV tosupport long term gene expression in transduced cells, to transduce bothdividing and nondividing cells, to transduce a wide variety of differenttypes of cells depending on serotype, the inability to replicate withouta helper virus, and an apparent lack of pathogenicity associated withwild type infections.

Because of their small size, AAV capsids can physically accommodate asingle stranded DNA genome that is at most about 4.7-5.0 kilobases inlength. Without modifying the genome, there would not be enough room toinclude a heterologous gene, such as coding sequence for a therapeuticprotein, and gene regulatory elements, such as a promoter and optionallyan enhancer. To create more room, the rep and cap genes can be removedand replaced with desired heterologous sequences, as long as theflanking ITRs are retained. The functions of the rep and cap genes canbe provided in trans on a different piece of DNA. By contrast, the ITRsare the only AAV viral elements that must remain in cis with theheterologous sequence. Combining the ITRs with a heterologous gene andremoving the rep and cap genes to a different plasmid lacking ITRs alsoprevents production of infectious wild type AAV at the same time thatAAV vector for gene therapy is being produced. Removing rep and cap alsomeans that AAV vectors for gene therapy cannot replicate in the cellsthey transduce.

In some embodiments, the genome of AAV vectors is linear single-strandedDNA flanked by AAV ITRs. Before it can support transcription andtranslation of the heterologous gene, the single stranded DNA genomemust be converted to double-stranded form by cellular DNA polymerasesthat utilize the free 3′-OH of one of the self-priming ITRs to initiatesecond-strand synthesis. In alternative embodiments, full length-singlestranded genomes of opposite polarity can anneal to generate a fulllength double-stranded genome, and can result when a plurality of AAVvectors carrying genomes of opposite polarity simultaneously transducethe same cell. After double-stranded vector genomes form, by whatevermechanism, the cellular gene transcription machinery can act on thedouble-stranded DNA to express the heterologous gene.

In other embodiments, the vector genome can be designed to beself-complementary (scAAV), having a wild type ITR at each end and amutated ITR in the middle. See, for example, McCarty, D M, et al.,Adeno-associated virus terminal repeat (TR) mutant generatesself-complementary vectors to overcome the rate-limiting step totransduction in vivo. Gene Ther. 10:2112-18 (2003). It has been proposedthat after entering a cell, self-complementary AAV genomes canself-anneal starting with the ITR in the middle to form adouble-stranded genome without need for de novo DNA replication. Thisapproach was shown to result in more efficient transduction and fasterexpression of heterologous gene, but reduces the size of theheterologous gene that may be used by about half.

Different strategies for producing AAV vectors for gene therapy havebeen developed, but one of the most common is the triple transfectiontechnique, in which three different plasmids are transfected intoproducer cells. See, for example, N. Clement and J. Grieger, Mol TherMethds Clin Dev, 3:16002 (2016), Grieger, J C, et al., Mol Ther24(2):287-97 (2016), and the references cited therein. In thistechnique, a plasmid is created that includes the sequence of the vectorgenome including, for example a heterologous promoter and optionally anenhancer, and a heterologous gene to express a desired RNA or protein,flanked by the left and right ITRs. The vector plasmid would beco-transfected into producer cells, such as HEK293 cells, with a secondplasmid containing the rep and cap genes, and a third plasmid containingadenovirus (or other virus) helper genes required to replicate andpackage the vector genome into AAV capsids. In alternative embodimentsof the technique, rep, cap and adenovirus helper genes all reside on thesame plasmid, and two plasmids are co-transfected into producer cells.Examples of adenovirus helper genes include E1a, E1b, E2a, E4orf6, andVA RNA genes. For many AAV serotypes, the AAV2 ITRs can be substitutedfor native ITRs without significantly impairing the ability of thevector genome to be replicated and packaged into non-AAV2 capsids. Thisapproach, known as pseudo-typing, merely requires using a rep/capplasmid that contains the rep and cap genes from the other serotype.Thus, for example, an AAV gene therapy vector could use an AAV9 capsidand a vector genome containing AAV2 ITRs flanking a heterologous gene(which can be designated “AAV2/9”), such as a mini-dystrophin. After theAAV particles are produced by the cell, they can be collected andpurified using standard techniques known in the art, such asultracentrifugation in a CsCl gradient, or using chromatography columnsof various types.

The parvovirus particles and genomes of the present invention can befrom, but are not limited to AAV. The genomic sequences of variousserotypes of AAV and the autonomous parvoviruses, as well as thesequences of the native ITRs, Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as GenBank. See, e.g., GenBank Accession NumbersNC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862,NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790,AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061,AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358,NC_001540, AF513851, AF513852 and AY530579; the disclosures of which areincorporated by reference herein for teaching parvovirus and AAV nucleicacid and amino acid sequences. See also, e.g., Bantel-Schaal et al.,(1999) J. Virol. 73: 939; Chiorini et al., (1997) J. Virol. 71:6823;Chiorini et al., (1999) J. Virol. 73:1309; Gao et al., (2002) Proc. Nat.Acad. Sci. USA 99:11854; Moris et al., (2004) Virol. 33-:375-383; Moriet al., (2004) Virol. 330:375; Muramatsu et al., (1996) Virol. 221:208;Ruffing et al., (1994) J. Gen. Virol. 75:3385; Rutledge et al., (1998)J. Virol. 72:309; Schmidt et al., (2008) J. Virol. 82:8911; Shade etal., (1986) J. Virol. 58:921; Srivastava et al., (1983) J Virol. 45:555;Xiao et al., (1999) J. Virol. 73:3994; international patent publicationsWO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303; thedisclosures of which are incorporated by reference herein for teachingparvovirus and AAV nucleic acid and amino acid sequences. ITR sequencesfrom AAV1, AAV2 and AAV3 are provided by Xiao, X., (1996),“Characterization of Adeno-associated virus (AAV) DNA replication andintegration,” Ph.D. Dissertation, University of Pittsburgh, Pittsburgh,PA (incorporated herein it its entirety).

As used herein, “transduction” of a cell by AAV refers to AAV-mediatedtransfer of genetic material into the cell. See, e.g., FIELDS et al.,VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

The terms “5′ portion” and “3′ portion” are relative terms to define aspatial relationship between two or more elements. Thus, for example, a“3′ portion” of a polynucleotide indicates a segment of thepolynucleotide that is downstream of another segment. The term “3′portion” is not intended to indicate that the segment is necessarily atthe 3′ end of the polynucleotide, or even that it is necessarily in the3′ half of the polynucleotide, although it may be. Likewise, a “5′portion” of a polynucleotide indicates a segment of the polynucleotidethat is upstream of another segment. The term “5′ portion” is notintended to indicate that the segment is necessarily at the 5′ end ofthe polynucleotide, or even that it is necessarily in the 5′ half of thepolynucleotide, although it may be.

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “polynucleotide” is a linear sequence of nucleotides in which the3′-position of each monomeric unit is linked to the 5′-position of theneighboring monomeric unit via a phosphate group. Polynucleotides may beRNA (containing RNA nucleotides only), DNA (containing DNA nucleotidesonly), RNA and DNA hybrids (containing RNA and DNA nucleotides), as wellas other hybrids containing naturally occurring and/or non-naturallyoccurring nucleotides. The linear order of bases of the nucleotides in apolynucleotide is called the “nucleotide sequence,” “nucleic acidsequence,” “nucleobase sequence,” or sometimes, just “sequence” of thepolynucleotide. Typically, the order of bases is provided starting fromthe 5′ end of the polynucleotide and ending at the 3′ end of thepolynucleotide. As known in the art, polynucleotides can adopt secondarystructures, such as regions of self-complementarity. Polynucleotides canalso hybridize with fully or partially complementary polynucleotidesthrough classic Watson-Crick base pairing, or other mechanisms familiarto those of ordinary skill.

As used herein, a “gene” is a section of a polynucleotide, typically butnot necessarily of DNA, that encodes a polypeptide or protein. In someembodiments, genes can be interrupted by introns. In some embodiments apolynucleotide can encode more than one polypeptide or protein due tomechanisms such as alternative splicing, use of alternate start codons,or other biological mechanisms familiar to those of ordinary skill inthe art. The term “open reading frame,” abbreviated “ORF,” refers to aportion of a polynucleotide that encodes a polypeptide or protein.

The term “codon-optimized,” as used herein, refers to a gene codingsequence that has been optimized to increase expression by substitutingone or more codons normally present in a coding sequence (for example,in a wildtype sequence, including, e.g., a coding sequence fordystrophin or a mini-dystrophin) with a codon for the same (synonymous)amino acid. In this manner, the protein encoded by the gene isidentical, but the underlying nucleobase sequence of the gene orcorresponding mRNA is different. In some embodiments, the optimizationsubstitutes one or more rare codons (that is, codons for tRNA that occurrelatively infrequently in cells from a particular species) withsynonymous codons that occur more frequently to improve the efficiencyof translation. For example, in human codon-optimization one or morecodons in a coding sequence are replaced by codons that occur morefrequently in human cells for the same amino acid. Codon optimizationcan also increase gene expression through other mechanisms that canimprove efficiency of transcription and/or translation. Strategiesinclude, without limitation, increasing total GC content (that is, thepercent of guanines and cytosines in the entire coding sequence),decreasing CpG content (that is, the number of CG or GC dinucleotides inthe coding sequence), removing cryptic splice donor or acceptor sites,and/or adding or removing ribosomal entry sites, such as Kozaksequences. Desirably, a codon-optimized gene exhibits improved proteinexpression, for example, the protein encoded thereby is expressed at adetectably greater level in a cell compared with the level of expressionof the protein provided by the wildtype gene in an otherwise similarcell.

The term “sequence identity,” as used herein, has the standard meaningin the art. As is known in the art, a number of different programs canbe used to identify whether a polynucleotide or polypeptide has sequenceidentity or similarity to a known sequence. Sequence identity orsimilarity may be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, WI), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387 (1984), preferably using the defaultsettings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5:151 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Meth. Enzymol., 266:460 (1996); blast.wustl/edu/blast/README.html.WU-BLAST-2 uses several search parameters, which are preferably set tothe default values. The parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucleic Acids Res. 25:3389 (1997).

A percentage amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

In a similar manner, percent nucleic acid sequence identity is definedas the percentage of nucleotide residues in the candidate sequence thatare identical with the nucleotides in the polynucleotide specificallydisclosed herein.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleotides than the polynucleotides specifically disclosedherein, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalnucleotides in relation to the total number of nucleotides. Thus, forexample, sequence identity of sequences shorter than a sequencespecifically disclosed herein, will be determined using the number ofnucleotides in the shorter sequence, in one embodiment. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as insertions, deletions,substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0,”which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

“Substantial homology” or “substantial similarity,” means, whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of thesequence.

As used herein, an “isolated” polynucleotide (e.g., an “isolated DNA” oran “isolated RNA”) means a polynucleotide separated or substantiallyfree from at least some of the other components of the naturallyoccurring organism or virus, for example, the cell or viral structuralcomponents or other polypeptides or nucleic acids commonly foundassociated with the polynucleotide.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

A “therapeutic polypeptide” is a polypeptide that may alleviate orreduce symptoms that result from an absence or defect in a protein in acell or subject. Alternatively, a “therapeutic polypeptide” is one thatotherwise confers a benefit to a subject, e.g., anti-cancer effects orimprovement in transplant survivability.

As used herein, the term “modified,” as applied to a polynucleotide orpolypeptide sequence, refers to a sequence that differs from a wild-typesequence due to one or more deletions, additions, substitutions, or anycombination thereof.

As used herein, by “isolate” or “purify” (or grammatical equivalents) avirus vector, it is meant that the virus vector is at least partiallyseparated from at least some of the other components in the startingmaterial.

By the terms “treat,” “treating,” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or stabilized and/orthat some alleviation, mitigation, decrease or stabilization in at leastone clinical symptom is achieved and/or there is a delay in theprogression of the disease or disorder.

The terms “prevent,” “preventing,” and “prevention” (and grammaticalvariations thereof) refer to prevention and/or delay of the onset of adisease, disorder and/or a clinical symptom(s) in a subject and/or areduction in the severity of the onset of the disease, disorder and/orclinical symptom(s) relative to what would occur in the absence of themethods of the invention. The prevention can be complete, e.g., thetotal absence of the disease, disorder and/or clinical symptom(s). Theprevention can also be partial, such that the occurrence of the disease,disorder and/or clinical symptom(s) in the subject and/or the severityof onset is less than what would occur in the absence of the presentinvention.

A “treatment effective” amount as used herein is an amount that issufficient to provide some improvement or benefit to the subject.Alternatively stated, a “treatment effective” amount is an amount thatwill provide some alleviation, mitigation, decrease or stabilization inat least one symptom in the subject. Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

A “prevention effective” amount as used herein is an amount that issufficient to prevent and/or delay the onset of a disease, disorderand/or clinical symptoms in a subject and/or to reduce and/or delay theseverity of the onset of a disease, disorder and/or clinical symptoms ina subject relative to what would occur in the absence of the methods ofthe invention. Those skilled in the art will appreciate that the levelof prevention need not be complete, as long as some benefit is providedto the subject.

The terms “heterologous” or “exogenous” nucleotide or nucleic acidsequence are used interchangeably herein and refer to a nucleic acidsequence that is not naturally occurring in the virus or a cell. In someembodiments, the heterologous nucleic acid comprises an open readingframe that encodes a polypeptide or nontranslated RNA of interest (e.g.,for delivery to a cell or subject).

As used herein, the terms “virus vector,” “viral vector,” “gene deliveryvector,” or sometimes just “vector,” refer to a virion or virus particlethat functions as a nucleic acid delivery vehicle and which comprises avector genome packaged within the virion or virus particle. Vectors canbe infectious or non-infectious. Non-infectious vectors cannot replicatethemselves without exogenously added factors. Vectors may be AAVparticles or virions comprising an AAV capsid within which is packagedan AAV vector genome. These vectors may also be referred to herein as“recombinant AAV” (abbreviated “rAAV”) vectors, particles or virions.

A vector genome is a polynucleotide for packaging within a vectorparticle or virion for delivery into a cell (which cell may be referredto as a “target cell”). Typically, a vector genome is engineered tocontain a heterologous nucleic acid sequence, such as a gene, fordelivery into the target cell. A vector genome may also contain one ormore nucleic acid sequences that function as regulatory elements tocontrol expression of the heterologous gene in the target cell. A vectorgenome may also contain wildtype or modified viral nucleic acidsequence(s) required for the production and/or function of the vector,such as, without limitation, replication of the vector genome in a hostand packaging into vector particles. In some embodiments, the vectorgenome is an “AAV vector genome,” which is capable of being packagedinto an AAV capsid. In some embodiments, an AAV vector genome includesone or two inverted terminal repeats (ITRs) in cis with the heterologousgene to support replication and packaging. All other structural andnon-structural protein coding sequences required for AAV vectorproduction may be provided in trans (e.g., from a plasmid, or by stablyintegrating the sequences into a host cell). In certain embodiments, anAAV vector genome comprises at least one ITR (e.g., an AAV ITR),optionally two ITRs (e.g., two AAV ITRs), which typically will be at the5′ and 3′ ends of the vector genome and flank the heterologous nucleicacid sequence, but need not be contiguous thereto. The ITRs can be thesame or different from each other, and from the same or different AAVserotypes.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants,” “transformed cells,” and “transducedcells,” which include the primary transformed cell and progeny derivedtherefrom without regard to the number of passages. For purposes ofproducing AAV vectors, certain host cells may be used as “producer” or“packaging” cells that contain all the genes required to assemblefunctional virus particles including a capsid and vector genome. Asunderstood by those of ordinary skill in the art, different host cellscan usefully serve as producer cells, such as HEK293 cells, or the Prol0cell line, but others are possible. The required genes for virionassembly include the vector genome as described elsewhere herein, AAVrep and cap genes, and certain helper genes from other viruses,including without limitation adenovirus. As appreciated by thoseordinarily skilled, the requisite genes for AAV production can beintroduced into producer cells in various ways, including withoutlimitation transfection of one or more plasmids, however, certain of thegenes can already be present in the producer cells, either integratedinto the genome or carried on an episome.

The term “inverted terminal repeat” or “ITR” includes any palindromicviral terminal repeat or synthetic sequence that forms a hairpinstructure and functions as an inverted terminal repeat (i.e., mediatescertain viral functions such as replication, virus packaging,integration and/or provirus rescue, and the like). The ITR can be an AAVITR or a non-AAV ITR. For example, a non-AAV ITR sequence such as thoseof other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouseparvovirus, porcine parvovirus, human parvovirus B-19) or the SV40hairpin that serves as the origin of SV40 replication can be used as anITR, which can further be modified by truncation, substitution,deletion, insertion and/or addition. Further, the ITR can be partiallyor completely synthetic, such as the “double-D sequence” as described inU.S. Pat. No. 5,478,745 to Samulski et al. See also FIELDS et al.,VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-RavenPublishers).

An “AAV inverted terminal repeat” or “AAV ITR” may be from any AAV,including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9,10, 11, or 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV,ovine AAV, goat AAV, shrimp AAV, or any other AAV now known or laterdiscovered. An AAV ITR need not have the native terminal repeat sequence(e.g., a native AAV ITR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,persistence, and/or provirus rescue, and the like. The sequence of theAAV2 ITRs are 145 basepairs long, and are provided herein as SEQ IDNO:14 and SEQ ID NO:15.

“Cis-motifs” includes conserved sequences such as found at or close tothe termini of the genomic sequence and recognized for initiation ofreplication; cryptic promoters or sequences at internal positions likelyused for transcription initiation, splicing or termination.

“Flanked,” with respect to a sequence that is flanked by other elements,indicates the presence of one or more the flanking elements upstreamand/or downstream, i.e., 5′ and/or 3′, relative to the sequence. Theterm “flanked” is not intended to indicate that the sequences arenecessarily contiguous. For example, there may be intervening sequencesbetween the nucleic acid encoding the transgene and a flanking element.A sequence (e.g., a transgene) that is “flanked” by two other elements(e.g., TRs), indicates that one element is located 5′ to the sequenceand the other is located 3′ to the sequence; however, there may beintervening sequences there between.

“Transfection” of a cell means that genetic material is introduced intoa cell for the purpose of genetically modifying the cell. Transfectioncan be accomplished by a variety of means known in the art, such ascalcium phosphate, polyethyleneimine, electroporation, and the like.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells. Such methods can resultin transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g. episomes), or integration of transferred genetic material into thegenomic DNA of host cells.

“Transgene” is used to mean any heterologous nucleotide sequenceincorporated in a vector, including a viral vector, for delivery to andincluding expression in a target cell (also referred to herein as a“host cell”), and associated expression control sequences, such aspromoters. It is appreciated by those of skill in the art thatexpression control sequences will be selected based on ability topromote expression of the transgene in the target cell. An example of atransgene is a nucleic acid encoding a therapeutic polypeptide.

The virus vectors of the invention can further be “targeted” virusvectors (e.g., having a directed tropism) and/or a “hybrid” parvovirus(i.e., in which the viral ITRs and viral capsid are from differentparvoviruses) as described in international patent publication WO00/28004 and Chao et al., (2000) Mol. Therapy 2:619.

Further, the viral capsid or genomic elements can contain othermodifications, including insertions, deletions and/or substitutions.

As used herein, parvovirus or AAV “Rep coding sequences” indicate thenucleic acid sequences that encode the parvoviral or AAV non-structuralproteins that mediate viral replication and the production of new virusparticles. The parvovirus and AAV replication genes and proteins havebeen described in, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 69& 70 (4th ed., Lippincott-Raven Publishers).

The “Rep coding sequences” need not encode all of the parvoviral or AAVRep proteins. For example, with respect to AAV, the Rep coding sequencesdo not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52and Rep40), in fact, it is believed that AAV5 only expresses the splicedRep68 and Rep40 proteins. In representative embodiments, the Rep codingsequences encode at least those replication proteins that are necessaryfor viral or vector genome replication and packaging into new virions.The Rep coding sequences will generally encode at least one large Repprotein (i.e., Rep78/68) and one small Rep protein (i.e., Rep52/40). Inparticular embodiments, the Rep coding sequences encode the AAV Rep78protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments,the Rep coding sequences encode the Rep68 and the Rep52 and/or Rep40proteins. In a still further embodiment, the Rep coding sequences encodethe Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52proteins, or Rep78 and Rep40 proteins.

As used herein, the term “large Rep protein” refers to Rep68 and/orRep78. Large Rep proteins of the claimed invention may be eitherwild-type or synthetic. A wild-type large Rep protein may be from anyparvovirus or AAV, including but not limited to serotypes 1, 2, 3a, 3b,4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or laterdiscovered. A synthetic large Rep protein may be altered by insertion,deletion, truncation and/or missense mutations.

In the native AAV genome, the different Rep proteins are encoded by asingle gene through use of two different promoters and alternativesplicing. For purposes of AAV vector production, however, Rep proteinscan be expressed in producer cells from a single gene, or from distinctpolynucleotides, one sequence for each Rep protein to be expressed.Thus, for example, a Rep encoding gene can be engineered to inactivatethe p5 or p19 promoter so that only small or only large Rep proteins areexpressed the respective modified genes. Expression of the large andsmall Rep proteins from different genes can be advantageous when one ofthe viral promoters is inactive in a host cell, in which case aconstitutively active promoter can be used instead, or where it isdesired to express the Rep proteins at different levels under thecontrol of separate transcriptional and/or translational controlelements. For example, in some embodiments, it may be advantageous todown-regulate expression of the large Rep protein relative to small Repprotein (e.g., Rep78/68) to avoid toxicity to the host cells (see, e.g.,Urabe et al., (2002) Human Gene Therapy 13:1935).

As used herein, the parvovirus or AAV “cap coding sequences” encode thestructural proteins that form a functional parvovirus or AAV capsid(i.e., can package DNA and infect target cells). Typically, the capcoding sequences will encode all of the parvovirus or AAV capsidsubunits, but less than all of the capsid subunits may be encoded aslong as a functional capsid is produced. Typically, but not necessarily,the cap coding sequences will be present on a single nucleic acidmolecule.

The capsid structure of autonomous parvoviruses and AAV are described inmore detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69& 70 (4th ed., Lippincott-Raven Publishers).

A “micro-dystrophin” or a “mini-dystrophin” is an engineered proteincomprising certain subdomains or portions of subdomains present in fulllength muscle dystrophin or isoforms thereof that possess at least someof the functionality of dystrophin when expressed in a muscle cell.Micro-dystrophins and mini-dystrophins are smaller than full lengthmuscle dystrophin (Dp427m). Relative to full length muscle dystrophin,micro-dystrophins and mini-dystrophins may contain deletions at theN-terminus, the C-terminus, internally, or any combination thereof.

As used herein, a “dystrophinopathy” is a muscle disease caused bypathogenic variants in DMD, the gene encoding the protein dystrophin.Dystrophinopathies manifest as a spectrum of phenotypes depending on thenature of the underlying genetic lesion. The mild end of the spectrumincludes without limitation the phenotypes of asymptomatic increase inserum concentration of creatine phosphokinase (CK) and muscle crampswith myoglobinuria. The severe end of the spectrum includes withoutlimitation the progressive muscle diseases Duchenne muscular dystrophy(DMD) and Becker muscular dystrophy (BMD), in which skeletal muscle isprimarily affected and heart to a lesser degree, and DMD-associateddilated cardiomyopathy (DCM), in which the heart is primarily affected.

Mini-Dystrophin Polynucleotides, Expression Cassettes and Vectors

The present disclosure provides codon-optimized mini-dystrophin genesequences and expression cassettes containing the same. Such genes andexpression cassettes are useful for, among other applications, genetherapy to prevent or treat dystrophinopathies, such as DMD, in subjectsin need thereof. Expression of mini-dystrophin proteins in transducedmuscle cells is able to replicate and replace at least some of thefunction normally attributable to full-length dystrophin, such assupporting a mechanically strong link between the extra-cellular matrixand the cytoskeleton.

The codon-optimized sequences are designed to fit within the sizelimitations of parvovirus vectors, e.g., AAV vectors, as well as provideenhanced expression of mini-dystrophin compared to non-optimizedsequences. In some embodiments, the optimized mini-dystrophin sequencesprovide increased expression of mini-dystrophin protein in muscle cellsor in muscle in animals that is at least about 5% greater than theexpression of non-codon-optimized dystrophin sequences, e.g., at leastabout 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or 500% or more,where the non-codon-optimized sequence is based on the mRNA encodingwildtype human full-length muscle dystrophin, as exemplified by NCBIReference Sequence NM_004006.2, which is incorporated by reference.

Thus, one aspect of the invention relates to a polynucleotide encoding amini-dystrophin protein, the polynucleotide comprising, consistingessentially of, or consisting of: (a) the nucleotide sequence of SEQ IDNO:1 or a sequence at least about 90% identical thereto; (b) thenucleotide sequence of SEQ ID NO:2 or a sequence at least about 90%identical thereto; or (c) the nucleotide sequence of SEQ ID NO:3 or asequence at least about 90% identical thereto. In some embodiments, thepolynucleotide is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the nucleotide sequence of one of SEQ ID NOS:1-3. In certain embodiments, the polynucleotide has a length that iswithin the capacity of a viral vector, e.g., a parvovirus vector, e.g.,an AAV vector. In some embodiments, the polynucleotide is about 5000,4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100, or about 4000nucleotides, or fewer.

In some embodiments, the mini-dystrophin protein encoded by thepolynucleotide comprises, consists essentially of, or consists of theN-terminus, hinge H1, rods R1 and R2, hinge H3, rods R22, R23, and R24,hinge H4, the cysteine-rich domain (CR domain), and in some embodiments,all or a portion of the carboxy-terminal domain (CT domain) of wild-typedystrophin protein. In other embodiments, the mini-dystrophin proteinencoded by the polynucleotide comprises, consists essentially of, orconsists of the N-terminus, Actin-Binding Domain (ABD), hinge H1, rodsR1 and R2, rods R22, R23, and R24, hinge H4, the CR domain, and in someembodiments, all or a portion of the CT domain of wild-type dystrophinprotein. In further embodiments, the mini-dystrophin protein does notcomprise the last three amino acids at the C-terminus of the wild-typedystrophin protein (SEQ ID NO:25). In certain embodiments, thepolynucleotide encodes a mini-dystrophin protein comprising, consistingessentially of, or consisting of the amino acid sequence of SEQ ID NO:7or SEQ ID NO:8 or a sequence at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQID NO:7 or SEQ ID NO:8.

The nucleotide sequence of dystrophin is well known in the art and maybe found in sequence databases such as GenBank. For example, the humandystrophin mRNA sequence may be found at GenBank Accession No. M18533 orNCBI Reference Sequence NM_004006.2, which are incorporated by referenceherein in their entirety.

In some embodiments, the polynucleotide is part of an expressioncassette for production of dystrophin protein. The expression cassettemay further comprise expression elements useful for increasingexpression of dystrophin.

In some embodiments, the polynucleotide of the invention is operablylinked to a promoter. The promoter may be a constitutive promoter or atissue-specific or tissue-preferred promoter such as a muscle-specificor muscle-preferred promoter. In some embodiments, the promoter is acreatinine kinase promoter, e.g., a promoter comprising, consistingessentially of, or consisting of the nucleotide sequence of SEQ ID NO: 4or SEQ ID NO: 5.

In some embodiments, the polynucleotide of the invention is operablylinked to a polyadenylation element. In some embodiments, thepolyadenylation element comprises the nucleotide sequence of SEQ ID NO:6.

In some embodiments, the polynucleotide is part of an expressioncassette comprising, consisting essentially of, or consisting or thepolynucleotide operably linked to a promoter and a polyadenylationelement. In certain embodiments, the gene expression cassette comprises,consists essentially or, or consists of the nucleotide sequence of anyone of SEQ ID NOS: 9-12 or a sequence at least about 90% identicalthereto, e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical.

Another aspect of the invention relates to a vector comprising thepolynucleotides of the invention. Suitable vectors include, but are notlimited to, a plasmid, phage, phagemid, viral vector (e.g., AAV vector,an adenovirus vector, a herpesvirus vector, an alphavirus, or abaculovirus vector), bacterial artificial chromosome (BAC), or yeastartificial chromosome (YAC). For example, the nucleic acid can comprise,consist of, or consist essentially of an AAV vector comprising a 5′and/or 3′ terminal repeat (e.g., 5′ and/or 3′ AAV terminal repeat). Insome embodiments, the vector is a viral vector, e.g., a parvovirusvector, e.g., an AAV vector, e.g., an AAV9 vector. The viral vector mayfurther comprise a nucleic acid comprising a recombinant viral template,wherein the nucleic acid is encapsidated by the parvovirus capsid. Theinvention further provides a recombinant parvovirus particle (e.g., arecombinant AAV particle) comprising the polynucleotides of theinvention. Viral vectors and viral particles are discussed furtherbelow.

In certain embodiments, the viral vector exhibits modified tissuetropism compared to vectors from which the modified vector is derived.In one embodiment, the parvovirus vector exhibits systemic tropism forskeletal, cardiac, and/or diaphragm muscle. In other embodiments, theparvovirus vector has reduced tropism for liver compared to a virusvector comprising a wild-type capsid protein. Tissue tropism can bemodified by altering certain viral capsid amino acids, for example,those present in AAV capsid VP1, VP2, and/or VP3 proteins, according tothe knowledge of those ordinarily skilled in the art.

In some embodiments, the vector genome is self-complementary orduplexed, and AAV virions containing such vector genomes are known asscAAV vectors. scAAV vectors are described in international patentpublication WO 01/92551 (the disclosure of which is incorporated hereinby reference in its entirety). Use of scAAV to express a mini-dystrophinmay provide an increase in the number of cells transduced, the copynumber per transduced cell, or both.

An additional aspect of the invention relates to a transformed cellcomprising the polynucleotide and/or vector of the invention. The cellmay be an in vitro, ex vivo, or in vivo cell.

A further aspect of the invention relates to a non-human transgenicanimal comprising the polynucleotide and/or vector and/or transformedcell of the invention. In some embodiments, the transgenic animal is alaboratory animal, e.g., an animal model of a disease, e.g., an animalmodel of muscular dystrophy.

Another aspect of the invention relates to a mini-dystrophin proteinencoded by the polynucleotides of the invention. The mini-dystrophinprotein contains all of the sequences necessary for a functionaldystrophin protein. The domains of dystrophin are well known in the artand sequences may be found in sequence databases such as GenBank. Forexample, the human dystrophin amino acid sequence may be found at NCBIReference Sequence: NP_003997.1 and GenBank Accession No. AAA53189,which are incorporated by reference herein in their entirety.

In some embodiments, the mini-dystrophin protein comprises, consistsessentially of, or consists of the N-terminus, hinge H1, rods R1 and R2,hinge H3, rods R22, R23, and R24, hinge H4, the CR domain, and in someembodiments, all or a portion of the CT domain, wherein themini-dystrophin protein does not comprise the last three amino acids atthe C-terminus of wild-type dystrophin protein (SEQ ID NO:25). Accordingto some of these embodiments, the N-terminal actin binding domaincomprises, consists essentially of, or consists of amino acid numbers1-240 from SEQ ID NO:25, the amino acid sequence of full length humandystrophin protein; H1 comprises, consists essentially of, or consistsof amino acid numbers 253-327 from SEQ ID NO:25; R1 comprises, consistsessentially of, or consists of amino acid numbers 337-447 from SEQ IDNO:25; R2 comprises, consists essentially of, or consists of amino acidnumbers 448-556 from SEQ ID NO:25; H3 comprises, consists essentiallyof, or consists of amino acid numbers 2424-2470 from SEQ ID NO:25; R22comprises, consists essentially of, or consists of amino acid numbers2687-2802 from SEQ ID NO:25; R23 comprises, consists essentially of, orconsists of amino acid numbers 2803-2931 from SEQ ID NO:25; R24comprises, consists essentially of, or consists of amino acid numbers2932-3040 from SEQ ID NO:25; H4 comprises, consists essentially of, orconsists of amino acid numbers 3041-3112 from SEQ ID NO:25; the CRdomain comprises, consists essentially of, or consists of amino acidnumbers 3113-3299 from SEQ ID NO:25; and the CT domain comprises,consists essentially of, or consists of amino acid numbers 3300-3408from SEQ ID NO:25. In certain embodiments, the mini-dystrophin proteincomprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 7. Further description of this and relatedconstructs is included in Example 1 herein.

In some embodiments, the mini-dystrophin protein comprises, consistsessentially of, or consists of the N-terminus, hinge H1, rods R1 and R2,rods R22, R23, and R24, hinge H4, the CR domain, and in someembodiments, all or a portion of the CT domain. In certain embodiments,the mini-dystrophin protein does not comprise the last three amino acidsat the C-terminus of wild-type dystrophin protein. According to some ofthese embodiments, the N-terminal actin binding domain comprises,consists essentially of, or consists of amino acid numbers 1-240 fromSEQ ID NO:25, the amino acid sequence of full length human dystrophinprotein; H1 comprises, consists essentially of, or consists of aminoacid numbers 253-327 from SEQ ID NO:25; R1 comprises, consistsessentially of, or consists of amino acid numbers 337-447 from SEQ IDNO:25; R2 comprises, consists essentially of, or consists of amino acidnumbers 448-556 from SEQ ID NO:25; R22 comprises, consists essentiallyof, or consists of amino acid numbers 2687-2802 from SEQ ID NO:25; R23comprises, consists essentially of, or consists of amino acid numbers2803-2931 from SEQ ID NO:25; R24 comprises, consists essentially of, orconsists of amino acid numbers 2932-3040 from SEQ ID NO:25; H4comprises, consists essentially of, or consists of amino acid numbers3041-3112 from SEQ ID NO:25; cysteine rich domain comprises, consistsessentially of, or consists of amino acid numbers 3113-3299 from SEQ IDNO:25; and carboxy-terminal domain comprises, consists essentially of,or consists of amino acid numbers 3300-3408 from SEQ ID NO:25. Incertain embodiments, the mini-dystrophin protein comprises, consistsessentially of, or consists of the amino acid sequence of SEQ ID NO: 8.

A further aspect of the invention relates to a method of producing amini-dystrophin protein in a cell, comprising contacting the cell withthe polynucleotide or vector of the invention, thereby producing themini-dystrophin in the cell. The cell may be an in vitro, ex vivo, or invivo cell, e.g., a cell line or a primary cell. Methods of producing aprotein in a cell by introduction of a polynucleotide encoding theprotein are well known in the art.

Another aspect of the invention relates to a method of producing amini-dystrophin protein in a subject, comprising delivering to thesubject the polynucleotide, vector and/or transformed cell of theinvention, thereby producing the mini-dystrophin protein in the subject.

An additional aspect of the invention relates to a method of treatingmuscular dystrophy in a subject in need thereof, comprising deliveringto the subject a therapeutically effective amount of the polynucleotide,vector, and/or transformed cell of the invention, thereby treatingmuscular dystrophy in the subject. The muscular dystrophy may be anyform of muscular dystrophy, e.g., Duchenne muscular dystrophy or Beckermuscular dystrophy.

Recombinant Virus Vectors

The virus vectors of the present invention are useful for the deliveryof polynucleotides encoding mini-dystrophin to cells in vitro, ex vivo,and in vivo. In particular, the virus vectors can be advantageouslyemployed to deliver or transfer polynucleotides encoding mini-dystrophinto animal, including mammalian, cells.

The virus vector may also comprise a heterologous nucleic acid thatshares homology with and recombines with a locus on a host chromosome.This approach can be utilized, for example, to correct a genetic defectin the host cell.

As a further alternative, the polynucleotides encoding mini-dystrophincan be used to produce mini-dystrophin protein in a cell in vitro, exvivo, or in vivo. For example, the virus vectors may be introduced intocultured cells and the expressed mini-dystrophin protein isolatedtherefrom.

It will be understood by those skilled in the art that thepolynucleotide encoding mini-dystrophin can be operably associated withappropriate control sequences. For example, the polynucleotide can beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, and/or enhancers, and the like.

Those skilled in the art will appreciate that a variety of promoter andoptionally enhancer elements can be used depending on the level andtissue-specific expression desired. The promoter/enhancer can beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Anenhancer, if employed, can be chosen from the same gene and species asthe promoter, from the orthologous gene in a different species as thepromoter, from a different gene in the same species as the promoter, orfrom a different gene in a different species as the promoter.

In particular embodiments, the promoter/enhancer elements can be nativeto the target cell or subject to be treated. In representativeembodiments, the promoters/enhancer element can be native to theheterologous nucleic acid sequence. The promoter/enhancer element isgenerally chosen so that it functions in the target cell(s) of interest.Further, in particular embodiments the promoter/enhancer element is amammalian promoter/enhancer element. The promoter/enhancer element maybe constitutive or inducible.

Inducible expression control elements are typically advantageous inthose applications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific or-preferred promoter/enhancer elements, and include muscle specific orpreferred (including cardiac, skeletal and/or smooth muscle specific orpreferred) promoter/enhancer elements. Other inducible promoter/enhancerelements include hormone-inducible and metal-inducible elements.Exemplary inducible promoters/enhancer elements include, but are notlimited to, a Tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametallothionein promoter.

In embodiments wherein the polynucleotide encoding mini-dystrophin istranscribed and then translated in the target cells, specific initiationsignals are generally included for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The virus vectors according to the present invention provide a means fordelivering polynucleotide encoding mini-dystrophin into a broad range ofcells, including dividing and non-dividing cells. The virus vectors canbe employed to deliver the polynucleotide to a cell in vitro, e.g., toproduce mini-dystrophin in vitro or for ex vivo gene therapy. The virusvectors are additionally useful in a method of delivering thepolynucleotide to a subject in need thereof, e.g., to expressmini-dystrophin. In this manner, the protein can be produced in vivo inthe subject. The subject can be in need of mini-dystrophin because thesubject has a deficiency of functional dystrophin. Further, the methodcan be practiced because the production of mini-dystrophin in thesubject may impart some beneficial effect.

The virus vectors can also be used to produce mini-dystrophin incultured cells or in a subject (e.g., using the subject as a bioreactorto produce the protein or to observe the effects of the protein on thesubject, for example, in connection with screening methods).

In general, the virus vectors of the present invention can be employedto deliver the polynucleotide encoding mini-dystrophin to treat and/orprevent any disease state for which it is beneficial to delivermini-dystrophin. Illustrative disease states include, but are notlimited to muscular dystrophies including Duchenne and Becker.

Virus vectors according to the instant invention find use in diagnosticand screening methods, whereby a polynucleotide encoding mini-dystrophinis transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model.

The virus vectors of the present invention can also be used for variousnon-therapeutic purposes, including but not limited to use in protocolsto assess gene targeting, clearance, transcription, translation, etc.,as would be apparent to one skilled in the art. The virus vectors canalso be used for the purpose of evaluating safety (spread, toxicity,immunogenicity, etc.). Such data, for example, are considered by theUnited States Food and Drug Administration as part of the regulatoryapproval process prior to evaluation of clinical efficacy.

According to certain embodiments of the disclosure of AAV vectors orparticles for treating dystrophinopathy, such as DMD, the disclosureprovides AAV vectors or particles including AAV capsids from an AAVserotype that has tropism for striated muscle, including withoutlimitation, skeletal muscle, including the diaphragm, and cardiacmuscle. Non-limiting examples of naturally occurring AAV capsids havingtropism for striated muscle are AAV1, AAV6, AAV7, AAV8, and AAV9.However, other embodiments include AAV capsids that are not known tooccur naturally, but rather have been engineered for the express purposeof creating novel AAV capsids that preferentially transduce striatedmuscle compared to other tissues. Such engineered capsids are known inthe art, but the disclosure encompasses new muscle-specific AAV capsidsyet to be developed. Non-limiting examples of muscle-specific engineeredAAV capsids were reported in Yu, C Y, et al., Gene Ther 16(8):953-62(2009), Asokan, A, et al., Nat Biotech 28(1):79-82 (2010 (describingAAV2i8), Bowles, D E, et al., Mol Therapy 20(2):443-455 (2012)(describing AAV 2.5), and Asokan, A, et al., Mol Ther 20(4):699-708(2012). The amino acid sequences of the capsid proteins, including VP1,VP2, and VP3 proteins, for many naturally and non-naturally occurringAAV serotypes are known in the art. In one non-limiting example, theamino acid sequence for the AAV9 serotype is provided as the amino acidsequence of SEQ ID NO:13.

The AAV particles of the disclosure for treating dystrophinopathy, suchas DMD, include a vector genome for expressing a mini-dystrophin proteinwith dystrophin subdomains selected to at least partially restore intransduced muscle cells the function supplied by the missing full lengthdystrophin protein. According to some embodiments, the mini-dystrophinprotein is constructed from subdomains from the full length wild typehuman dystrophin protein. In some embodiments, the mini-dystrophinprotein includes the following subdomains from the human dystrophinprotein in the following order from N-terminus to C-terminus: N-terminalactin binding domain (ABD); H1 hinge domain; R1 and R2 spectrin-likerepeat domains; H3 hinge domain; R22, R23 and R24 spectrin-like repeatdomains; H4 hinge domain; cysteine rich (CR) domain; andcarboxy-terminal (CT) domain. According to some of these embodiments,the N-terminal actin binding domain comprises, consists essentially of,or consists of amino acid numbers 1-240 from SEQ ID NO:25, the aminoacid sequence of full length human dystrophin protein; H1 comprises,consists essentially of, or consists of amino acid numbers 253-327 fromSEQ ID NO:25; R1 comprises, consists essentially of, or consists ofamino acid numbers 337-447 from SEQ ID NO:25; R2 comprises, consistsessentially of, or consists of amino acid numbers 448-556 from SEQ IDNO:25; H3 comprises, consists essentially of, or consists of amino acidnumbers 2424-2470 from SEQ ID NO:25; R22 comprises, consists essentiallyof, or consists of amino acid numbers 2687-2802 from SEQ ID NO:25; R23comprises, consists essentially of, or consists of amino acid numbers2803-2931 from SEQ ID NO:25; R24 comprises, consists essentially of, orconsists of amino acid numbers 2932-3040 from SEQ ID NO:25; H4comprises, consists essentially of, or consists of amino acid numbers3041-3112 from SEQ ID NO:25; the CR domain comprises, consistsessentially of, or consists of amino acid numbers 3113-3299 from SEQ IDNO:25; and the CT domain comprises, consists essentially of, or consistsof amino acid numbers 3300-3408 from SEQ ID NO:25. According to certainembodiments, the mini-dystrophin protein has the amino acid sequence ofSEQ ID NO:7.

The vector genome of the AAV particles of the disclosure for treatingdystrophinopathy, such as DMD, includes a gene for expressing amini-dystrophin. Typically, the vector genome will lack the rep and capgenes normally present in wild type AAV to provide room for the geneexpressing the mini-dystrophin. In some embodiments, the gene encodes amini-dystrophin protein with the following subdomains from full lengthhuman dystrophin protein: ABD-H1-R1-R2-H3-R22-R23-R24-H4-CRD-CTD. Insome embodiments, the CTD is only a portion of the CTD found in wildtypemuscle dystrophin, and in some embodiments does not include the lastthree amino acids present in wildtype muscle dystrophin (SEQ ID NO:25).In certain embodiments, the gene encodes for a human mini-dystrophinprotein having the amino acid sequence of SEQ ID NO:7.

According to some embodiments, the gene encoding the humanmini-dystrophin protein is codon-optimized with respect to the speciesof the subject to which the AAV particles of the disclosure will beadministered to effect gene therapy. Without wishing to be bound bytheory, it is believed that codon-optimization improves the efficiencywith which transduced cells are able to transcribe the gene into mRNAand/or translate the mRNA into protein, thereby increasing the amount ofmini-dystrophin protein produced compared to expression of amini-dystrophin encoding gene that is non-codon-optimized. In somenon-limiting embodiments, the codon-optimization is humancodon-optimization, but codon-optimization can be performed with respectto other species, including canine.

In some embodiments, codon-optimization substitutes one or more codonsthat pair with relatively rare tRNAs present in a species, such ashuman, with synonymous codons that pair with more prevalent tRNAs forthe same amino acid. This approach can increase the efficiency oftranslation. In other embodiments, codon-optimization eliminates certaincis-acting motifs that can influence the efficiency of transcription ortranslation. Non-limiting examples of codon-optimization include addinga strong Kozak sequence at the intended start of the coding sequence, oreliminating internal ribosome entry sites downstream of the intendedstart codon. Other cis-acting motifs that may be eliminated throughcodon-optimization include internal TATA-boxes; chi-sites; ARE, INS,and/or CRS sequence elements; repeat sequences and/or RNA secondarystructures; cryptic splice donor and/or acceptor sites, branch points;and SalI sites.

In certain embodiments, codon-optimization increases the GC content(that is, the number of G and C nucleobases present in a nucleic acidsequence, usually expressed as a percentage) relative to the wildtypesequence from which the mini-dystrophin gene was assembled. In someembodiments, the GC content is at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or greater than the GC content of the corresponding wildtype gene. Inrelated embodiments, the GC content of a codon-optimized gene is aboutor at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, orgreater.

In some embodiments, codon-optimization increases the codon adaptationindex (CAI) of the gene encoding the mini-dystrophin protein. The CAI isa measure of synonymous codon usage bias in a particular species. TheCAI value (which ranges from 0 to 1) in a particular species ispositively correlated with gene expression levels. See, for example,Sharp, PM and W-H Lie, Nuc Acids Res 15(3):1281-95 (1987). According tocertain embodiments, codon-optimization increases the CAI of themini-dystrophin gene in reference to highly expressed human genes to avalue that is at least 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77,0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89,0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

In other embodiments, codon-optimization reduces the number of CpGdinucleotides in the coding sequence of a mini-dystrophin. Withoutwishing to be bound by any particular theory of operation, it isbelieved that methylation at CpG dinucleotides can silence genetranscription, such that reducing the number of CpG dinucleotides in agene sequence can reduce the level of methylation, thereby resulting inenhanced transcription efficiency. Thus, in some embodiments of thecodon-optimized mini-dystrophin genes, the number of CpG dinucleotidesis reduced by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, or more compared to the wildtype sequence from whichthe mini-dystrophin gene was assembled.

A non-limiting example of a human codon-optimized human mini-dystrophingene is provided by the DNA sequence of SEQ ID NO:1. This DNA sequence,which is 3978 nucleobases long (including a stop codon) is referred toherein as Hopti-Dys3978, although the particular terminology is merelyused for convenience and is not intended to be limiting. Themini-dystrophin protein sequence encoded by SEQ ID NO:1, which is calledDys3978, is provided by SEQ ID NO:7. An example of a caninecodon-optimized human mini-dystrophin gene is provided by SEQ ID NO:3,which also encodes Dys3978. As described in additional detail herein,the coding sequence for the mini-dystrophin of SEQ ID NO:7 was assembledfrom subsequences of the wildtype full-length human muscle dystrophingene (as exemplified by NCBI Reference Sequence NM_004006.2, which isincorporated by reference) corresponding to certain subdomains presentin the dystrophin protein (SEQ ID NO:25). The resulting gene sequence isprovided herein as SEQ ID NO:26, which was then human codon-optimized,resulting in the DNA sequence of SEQ ID NO:1. Without limitation, thecodon-optimization increased the GC content, decreased the use ofinfrequent codons (that is, increased the codon-adaptation index (CAI)),and included a strong translation initiation site (Kozak consensussequence or similar), compared to the gene sequence beforecodon-optimization.

The vector genome of the AAV particles of the disclosure for treatingdystrophinopathy, such as DMD, further include AAV inverted terminalrepeats (ITR) flanking the codon-optimized gene encoding mini-dystrophinprotein. In some embodiments, the ITRs are from the same AAV serotype asthe capsid (for example, without limitation AAV9 ITRs used with AAV9capsid), but in other embodiments, AAV ITRs from a different serotypemay be used. For example, ITRs from the AAV2 serotype may be used in avector genome in combination with an AAV capsid from a different,non-AAV2 serotype. Non-limiting examples include use of AAV2 ITRs with acapsid from the AAV1, AAV6, AAV7, AAV8, or AAV9 serotypes, or adifferent naturally or non-naturally occurring AAV serotype. In aparticular non-limiting example, AAV2 ITRs may be used in combinationwith the capsid from the AAV9 serotype. From the perspective of the plusor sense DNA strand of the vector genome, the sequence of the left, 5′,or upstream AAV2 ITR is provided as the DNA sequence of SEQ ID NO:14,and the sequence of the right, 3′, or downstream AAV2 ITR is provided asthe DNA sequence of SEQ ID NO:15.

The vector genome of the AAV vectors of the disclosure for treatingdystrophinopathy, such as DMD, further includes a transcriptionalregulatory element operably linked with the gene encoding themini-dystrophin protein so that the vector genome, once converted intoits double stranded form can express the mini-dystrophin gene intransduced cells. Transcriptional regulatory elements typically includea promoter, but optionally one or more enhancer elements that can act toaugment the rate of transcription initiation from the promoter.

Operable linkage of a transcriptional regulatory element with respect tothe mini-dystrophin coding sequence means that the transcriptionalregulatory element can function to control transcription and expressionof the gene, but does not necessarily require any particular structuralor spatial relationship. Because vector genomes of the disclosure aretypically packaged into AAV capsids as single-stranded DNA molecules, itshould be understood that the operable linkage may not be functionaluntil the vector genome is converted into double-stranded form. Usually,a promoter will be positioned 5′ or upstream of a gene sequence encodingthe mini-dystrophin protein, but other transcriptional regulatoryelements, such as enhancers, may be positioned 5′ or elsewhere, such as3′, of the gene.

In some embodiments, the transcriptional regulatory element can be astrong constitutively active promoter, such those found in certainviruses that infect eukaryotic cells. A well-known example from the artinclude the promoter from the cytomegalovirus (CMV), but others areknown as well such at the promoter from the Rous sarcoma virus (RSV).Strong viral promoters such as CMV or RSV are typically not tissuespecific, so that if used the mini-dystrophin protein would be expressednot only in muscle cells, but any other cell type, such as liver,transduced by the AAV particles of the disclosure. Hence, in otherembodiments, a muscle-specific transcriptional regulatory element can beused to reduce the amount of mini-dystrophin protein expressed innon-muscle cells, such as liver cells, that may also be transduced bythe AAV particles of the disclosure.

Muscle-specific transcriptional regulatory elements can be derived frommuscle-specific genes from any species, including mammalian species,such as without limitation, human or mouse muscle genes. Muscle-specifictranscriptional regulatory elements will typically include at minimum apromoter from a muscle-specific gene as well as one or more enhancersfrom the same or a different muscle specific gene. Such enhancers canoriginate from many parts of the native gene, such as enhancerspositioned 5′ or 3′ of the gene, or even reside in introns.Muscle-specific transcriptional regulatory elements can be removed enbloc from a muscle-specific gene and inserted into a plasmid forproducing the AAV vector genomes of the disclosure, or can be engineeredto tailor their activity and reduce their size as much as possible.

Non-limiting examples of muscle-specific genes from whichmuscle-specific transcriptional regulatory elements can be derivedinclude the muscle creatine kinase gene, myosin heavy chain gene, ormyosin light chain gene, or the alpha 1 actin gene from skeletal muscle,though others are possible as well. These genes can be from human,mouse, or other species.

Muscle-specific transcriptional regulatory elements that have beencreated for use in gene therapy applications are described in the art,and may be used in the AAV vectors of the disclosure for treatingmuscular dystrophy. In non-limiting examples, Hauser describedmuscle-specific transcriptional regulatory elements known as CK4, CK5,and CK6 derived from the mouse creatine kinase (MCK) gene (Hauser, M A,et al., Mol Therapy 2(1):16-25 (2000)), Salva described muscle-specifictranscriptional regulatory elements known as CK1 and CK7, derived fromthe MCK gene, and MHCK1 and MHCK7, which additionally include enhancersfrom the mouse α-MHC gene (Salva, M Z, et al., Mol Therapy 15(2):320-9(2007)), and Wang described muscle-specific transcriptional regulatoryelements known as enh358MCK, dMCK and tMCK (Wang, B, et al., GeneTherapy 15:1489-9 (2008)). Use of other muscle-specific transcriptionalregulatory elements in the AAV vectors of the disclosure for treatingmuscular dystrophy are also possible.

Non-limiting examples of muscle-specific transcriptional regulatoryelements that may be used in the AAV vectors of the disclosure fortreating muscular dystrophy include CK4, CK5, CK6, CK1, CK7, MHCK1,MHCK7, enh358MCK, dMCK and tMCK, each as described in the art, or thosedisclosed herein as having the DNA sequences of SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:16. Other muscle-specific transcriptional regulatoryelements may be used as well.

The vector genome of the AAV vectors of the disclosure for treatingdystrophinopathy, such as DMD, further includes a transcriptiontermination sequence positioned 3′ of the coding sequence for themini-dystrophin gene. Inclusion of transcription termination sequenceensures that the mRNA transcript encoding the mini-dystrophin proteinwill be appropriately polyadenylated by the transduced cell therebyensuring efficient translation of the message into protein. Withoutintending to be limited by any particular theory of operation, researchinto mammalian transcription termination sequences identified aconsensus sequence in the 3′ UTR of genes that serves to terminatetranscription and signal polyadenylation of the growing transcript.Specifically, these sequences typically include the motif AATAAA,followed by 15-30 nucleotides, and then CA. See, for example, N.Proudfoot, Genes Dev 25:1770-82 (2011). Other motifs, such as anupstream element (USE) and downstream element (DSE) may contribute totranscription termination in some genes. Many transcription terminationsequences are known in the art and can be used in the AAV vectors of thedisclosure. Non-limiting examples include the polyadenylation signalfrom the SV40 virus early or late genes (SV40 early or late polyA) orthe polyadenylation signal from the bovine growth hormone gene (bGHpolyA). Transcription termination sequences from other genes of anyspecies may be used in the AAV vectors of the disclosure. Alternatively,synthetic transcription termination sequences may be designed and usedto signal transcription termination and polyadenylation. Additionalnon-limiting examples of transcription termination sequences that may beused in the AAV vectors of the disclosure include those disclosed hereinas having the DNA sequences of SEQ ID NO:6 and SEQ ID NO:17.

According to certain non-limiting embodiments, the disclosure providesan AAV viral particle or vector for treating dystrophinopathy, such asDMD, comprising an AAV capsid and a vector genome encoding amini-dystrophin protein. In some embodiments, the mini-dystrophinprotein includes the following subdomains from full length humandystrophin protein: ABD-H1-R1-R2-H3-R22-R23-R24-H4-CRD-CTD. In someembodiments, the CTD is only a portion of the CTD found in wildtypemuscle dystrophin, and in some embodiments does not include the lastthree amino acids present in wildtype muscle dystrophin (SEQ ID NO:25).According to certain embodiments, the gene encoding the mini-dystrophinprotein of SEQ ID NO:7 is human codon-optimized and has the DNA sequenceof SEQ ID NO:1. In some embodiments, the AAV capsid is from the AAV9serotype.

As noted elsewhere herein, single-stranded AAV vector genomes arepackaged into capsids as the plus strand or minus strand in about equalproportions. Consequently, embodiments of the vector or particle includeAAV particles in which the vector genome is in the plus strand polarity(that is, has the nucleobase sequence of the sense or coding DNAstrand), as well as AAV particles in which the vector genome is in theminus strand polarity (that is, has the nucleobase sequence of theantisense or template DNA strand). Given the nucleobase sequence of theplus strand in its regular 5′ to 3′ order, the nucleobase sequence ofthe minus strand in its 5′ to 3′ order can be determined as thereverse-complement of the nucleobase sequence of the plus strand.

In some embodiments of the vector, the vector genome, when in pluspolarity, comprises a muscle-specific transcriptional regulatory elementderived from the creatine kinase gene having the DNA sequence of SEQ IDNO:16 positioned 5′ of and operably linked with SEQ ID NO:1, the DNAsequence of the human codon-optimized gene encoding mini-dystrophinprotein. Particles comprising the corresponding minus strand are alsopossible, where the sequence of nucleobases from its 5′ end would be thereverse complement of the sequence of the aforementioned plus strand. Inother embodiments, the vector genome, when in plus polarity comprises afirst AAV2 ITR followed by the DNA sequence of SEQ ID NO:16 positioned5′ of and operably linked with the DNA sequence of SEQ ID NO:1, and atranscription termination sequence comprising the DNA sequence of SEQ IDNO:17 positioned 3′ of the mini-dystrophin gene, followed by a secondAAV2 ITR. Particles comprising the corresponding minus strand are alsopossible, where the sequence of nucleobases from its 5′ end would be thereverse complement of the sequence of the aforementioned plus strand.

In certain other embodiments of the vector, the vector genome, when inplus polarity, comprises in 5′ to 3′ order a first AAV2 ITR, atranscriptional regulatory element sequence defined by the DNA sequenceof SEQ ID NO:16, a human codon optimized gene sequence for expressing amini-dystrophin, the gene sequence defined by the DNA sequence of SEQ IDNO:1 in operable linkage with the transcriptional regulatory element, atranscription termination sequence defined by the DNA sequence of SEQ IDNO:17, and a second AAV2 ITR. Particles comprising the correspondingminus strand are also possible, where the sequence of nucleobases fromits 5′ end would be the reverse complement of the sequence of theaforementioned plus strand.

According to a particular non-limiting embodiment, an AAV vector fortreating dystrophinopathy, such as DMD, which may be referred to hereinas AAV9.hCK.Hopti-Dys3978.spA, comprises a capsid from the AAV9 serotypeand a vector genome, which vector genome may be referred to herein ashCK.Hopti-Dys3978.spA, comprising, consisting essentially of, orconsisting of, when the genome is in plus polarity, the DNA sequence ofSEQ ID NO:18 or, when the genome is in the minus polarity, thereverse-complement of the DNA sequence of SEQ ID NO:18 (that is, whenthe vector genome sequence is read 5′ to 3′).

Methods of Producing Virus Vectors

The present disclosure further provides methods of producing AAVvectors. In one particular embodiment, the present disclosure provides amethod of producing a recombinant parvovirus particle, comprisingproviding to a cell permissive for AAV replication and packaging arecombinant AAV vector genome, comprising a mini-dystrophin gene,associated genetic control elements and flanking AAV ITRs, and AAVreplication and packaging functions, such as those provided by the AAVrep and cap genes, under conditions sufficient for the replication andpackaging of the recombinant AAV particles, whereby rAAV particles areproduced by the cell. Conditions sufficient for the replication andpackaging of the rAAV particles include without limitation helperfunctions, such as those from adenovirus and/or herpesvirus. Cellspermissive for AAV replication and packaging are known herein aspackaging cells or producer cells, terms encompassed by the broader termhost cells. The rAAV particle vector genome, replication and packagingfunctions and, where required, helper functions can be provided viaviral or non-viral vectors, such as plasmids, and can exist within thepackaging cells stably or transiently, either integrated into the cell'sgenome or in an episome.

Recombinant AAV vectors of the disclosure can be made by several methodsknown to skilled artisans (see, e.g., WO 2013/063379). An exemplarymethod is described in Grieger, et al. 2015, Molecular Therapy24(2):287-297, the contents of which are incorporated by reference.Briefly, efficient transfection of HEK293 cells is used as a startingpoint, wherein an adherent HEK293 cell line from a qualified clinicalmaster cell bank is used to grow in animal component-free suspensionconditions in shaker flasks and WAVE bioreactors that allow for rapidand scalable rAAV particle production. Using the triple transfectionmethod (e.g., WO 96/40240), the suspension HEK293 cell line is capableof generating, in some embodiments, greater than 1×10⁵ vector genome(vg) containing particles per cell, or greater than 1×10¹⁴ vg/L of cellculture when harvested 48 hours post-transfection. Triple transfectionrefers to the fact that the packaging cell is transfected with threeplasmids: one plasmid encodes the AAV rep and cap genes, another plasmidencodes various helper functions (e.g., adenovirus or HSV proteins suchas Ela, E1b, E2a, E4, and VA RNA, and another plasmid encodes the vectorgenome, i.e., the mini-dystrophin gene and its various control elementsflanked by AAV ITRs. To achieve the desired yields, a number ofvariables can be optimized such as selection of a compatible serum-freesuspension media that supports both growth and transfection, selectionof a transfection reagent, transfection conditions and cell density.Vectors can be collected from the medium and/or by lysing the cells, andthen purified using the classic density gradient ultracentrifugationtechnique, or using column chromatographic or other techniques.

The packaging functions include genes for viral vector replication andpackaging. Thus, for example, the packaging functions may include, asneeded, functions necessary for viral gene expression, viral vectorreplication, rescue of the viral vector from the integrated state, viralgene expression, and packaging of the viral vector into a viralparticle. The packaging functions may be supplied together or separatelyto the packaging cell using a genetic construct such as a plasmid or anamplicon, a Baculovirus, or HSV helper construct. The packagingfunctions may exist extrachromosomally within the packaging cell, butmay also be integrated into the cell's chromosomal DNA. Examples includegenes encoding AAV Rep and Cap proteins. Rep and cap genes can beprovided to packaging cell together as part of the same viral ornon-viral vector. For example, the rep and cap sequences may be providedby a hybrid adenovirus vector (e.g., inserted into the Ela or E3 regionsof a deleted adenovirus vector) or herpesvirus vector, such as an EBVvector. Alternatively, AAV rep and cap genes can be provided separately.Rep and cap genes can also be stably integrated into the genome of apackaging cell, or exist on an episome. Typically, rep and cap geneswill not be flanked by ITRs to avoid packaging of these sequences intorAAV vector particles.

The helper functions include helper virus elements needed forestablishing active infection of the packaging cell which is required toinitiate packaging of the viral vector. Examples include functionsderived from adenovirus, baculovirus and/or herpes virus sufficient toresult in packaging of the viral vector. For example, adenovirus helperfunctions will typically include adenovirus components Ela, E1b, E2a,E4, and VA RNA. The packaging functions may be supplied by infection ofthe packaging cell with the required virus. Alternatively, use ofinfectious virus can be avoided, whereby the packaging functions may besupplied together or separately to the packaging cell using a non-viralvector such as a plasmid or an amplicon. See, e.g., pXR helper plasmidsas described in Rabinowitz et al., 2002, J. Virol. 76:791, and pDGplasmids described in Grimm et al., 1998, Human Gene Therapy9:2745-2760. The packaging functions may exist extrachromosomally withinthe packaging cell, but may also be integrated into the cell'schromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

Any method of introducing the nucleotide sequence carrying the helperfunctions into a cellular host for replication and packaging may beemployed, including but not limited to electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the helper functions are provided by transfectionusing a virus vector or infection using a helper virus; standard methodsfor producing viral infection may be used.

Any suitable permissive or packaging cell known in the art may beemployed in the production of the packaged viral vector. Mammalian cellsor insect cells are preferred. Examples of cells useful for theproduction of packaging cells in the practice of the invention include,for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK 293cells (which express functional adenoviral E1 under the control of aconstitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2,HuH7, and HT1080 cell lines. In one aspect, the packaging cell iscapable of growing in suspension culture, especially in serum-freegrowth media. In one embodiment, the packaging cell is a HEK293 thatgrows in suspension in serum free medium. In another embodiment, thepackaging cell is the HEK293 cell described in U.S. Pat. No. 9,441,206and deposited as ATCC No. PTA 13274. Numerous rAAV particle packagingcell lines are known in the art, including, but not limited to, thosedisclosed in WO 2002/46359.

Cell lines for use as packaging cells include insect cell lines,particularly when baculoviral vectors are used to introduce the genesrequired for rAAV particle production as described herein. Any insectcell that allows for replication of AAV and that can be maintained inculture can be used in accordance with the present disclosure. Examplesinclude Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines,Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedesalbopictus-derived cell lines.

After AAV vector particles of the disclosure have been produced andpurified, they can be titered to prepare compositions for administrationto subjects, such as human subjects with muscular dystrophy. AAV vectortitering can be accomplished using methods known in the art. In certainembodiments, AAV vector particles can be titered using quantitative PCR(qPCR) using primers against sequences in the vector genome, forexample, AAV2 ITR sequences if present, or other sequences in the vectorgenome. By performing qPCR in parallel on dilutions of a standard ofknown concentration, such as a plasmid containing the sequence of thevector genome, a standard curve can be generated permitting theconcentration of the AAV vector to be calculated as the number of vectorgenomes (vg) per unit volume, such as microliters or milliliters.Alternatively, the number of AAV vector particles containing genomes canbe determined using dot blot using a suitable probe for the vectorgenome. These techniques are described further in Gray, S J, et al.,Production of recombinant adeno-associated viral vectors and use in invitro and in vivo administration, Curr Protoc Neurosci (2011) andWerling N J, et al., Gene Ther Meth 26:82-92 (2015). Once theconcentration of AAV vector genomes in the stock is determined, it canbe diluted into or dialyzed against suitable buffers for use inpreparing a composition for administration to subjects.

Methods of Treatment

The disclosure provides methods for treating a dystrophinopathy byadministering to a subject in need of treatment for dystrophinopathy atherapeutically effective dose or amount of an AAV vector of thedisclosure, such as, without limitation, the vector known asAAV9.hCK.Hopti-Dys3978.spA. In some embodiments, the dystrophinopathy isa muscular dystrophy, including without limitation Duchenne musculardystrophy (DMD), Becker muscular dystrophy (BMD), DMD-associated dilatedcardiomyopathy (DCM), and symptomatic carrier states in females. Thus,in some embodiments, the disclosure provides methods for treatingmuscular dystrophy by administering to a subject in need of treatmentfor muscular dystrophy a therapeutically effective dose or amount of anAAV vector of the disclosure, such as, without limitation, the vectorknown as AAV9.hCK.Hopti-Dys3978.spA. In related embodiments, thedisclosure provides methods for treating Duchenne muscular dystrophy(DMD), Becker muscular dystrophy (BMD), DMD-associated dilatedcardiomyopathy (DCM), and symptomatic carrier states in females, insubjects in need of treatment therefore.

Also provided is the use of an AAV vector or pharmaceutical compositionof the disclosure in the manufacture of a medicament for use in themethods of treatment disclosed herein. In addition, there is provided anAAV vector or pharmaceutical composition of the disclosure for use in amethod of treatment disclosed herein.

Treatment of subjects with a dystrophinopathy, such as DMD, need notresult in a cure to be considered effective, where cure is defined aseither halting disease progression, or partially or completely restoringthe subject's muscle function. Rather a therapeutically effective doseor amount of an AAV vector of the disclosure is one that serves toreduce or ameliorate the symptoms of, slow the progression of, orimprove the quality of life of a subject with the dystrophinopathy, suchas DMD. According to certain non-limiting embodiments, treatment ofsubjects with a dystrophinopathy can improve their mobility, delay thetime to their loss of ambulation or other mobility, and in the cases ofsevere dystrophinopathy, such as DMD, extend the life of subjects withthe disorder.

The methods of treatment of the disclosure can be used to treat male orfemale subjects with a dystrophinopathy, such as DMD. In the case offemales, treatment can be provided to symptomatic carriers, or to therare female subject with full blown disease. The methods of thedisclosure can also be used to treat subjects of any age with adystrophinopathy, including subjects less than 1 year old, or about orat least 1 year old, or about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 years old or older. Subjects, when treated, may be ambulatory, ornon-ambulatory.

The methods of treatment of the disclosure can be used to treat subjectswith a dystrophinopathy regardless of the underlying genetic lesion (forexample, deletions, duplications, splice site variants, or nonsensemutations in the dystrophin gene), so long as the lesion results in areduction or loss in the function of the native human dystrophin gene.

In certain embodiments of the disclosure, treating a subject with atherapeutically effective dose or amount of an AAV mini-dystrophinvector will reduce tissue concentrations of one or more biomarkers thatare associated with the existence or progression of muscular dystrophy.

According to certain embodiments, the biomarkers are certain enzymesreleased from damaged skeletal muscle or cardiac muscle cells into theblood (including serum or plasma). Non-limiting examples includecreatinine kinase (CK), the transaminases alanine aminotransferase (ALT)and aspartate aminotransferase (AST), and lactic acid dehydrogenase(LDH), the average levels of which are all known to be elevated insubjects with DMD.

In some embodiments, a therapeutically effective dose or amount of anAAV mini-dystrophin vector of the disclosure is effective to reduceelevated ALT levels in blood of DMD patients to within about 7-, 6-, 5-,4-, 3-, or 2-fold greater than that typically found in healthy subjectsof similar age and sex. In other embodiments, a therapeuticallyeffective dose or amount of an AAV mini-dystrophin vector of thedisclosure is effective to reduce elevated AST levels in blood of DMDpatients to within about 7-, 6-, 5-, 4-, 3-, or 2-fold greater than thattypically found in healthy subjects of similar age and sex. In someembodiments, a therapeutically effective dose or amount of an AAVmini-dystrophin vector of the disclosure is effective to reduce elevatedLDH levels in blood of DMD patients to within about 7-, 6-, 5-, 4-, 3-,or 2-fold greater than that typically found in healthy subjects ofsimilar age and sex. And in some other embodiments, a therapeuticallyeffective dose or amount of an AAV mini-dystrophin vector of thedisclosure is effective to reduce elevated total CK levels in blood ofDMD patients to within about 50-, 48-, 46-, 44-, 42-, 40-, 38-, 36-,34-, 32-, 30-, 28-, 26-, 24-, 22-, 20-, 18-, 16-, 14-, 12-, 10-, 9-, 8-,7-, 6-, 5-, 4-, 3-, or 2-fold greater than that typically found inhealthy subjects of similar age and sex. It has also been found thatmatrix metalloproteinase-9 (MMP-9), an enzyme associated withdegradation or remodeling of the extracellular matrix, is elevated inthe blood of DMD patients. See, for example, Nadaraja, V D, et al.,Neuromusc. Disorders 21:569-578 (2011). Thus, in some embodiments, atherapeutically effective dose or amount of an AAV mini-dystrophinvector of the disclosure is effective to reduce elevated MMP-9 levels inblood of DMD patients to within about 15-, 14-, 13-, 12-, 11-, 10-, 9-,8-, 7-, 6-, 5-, 4-, 3-, or 2-fold greater than that typically found inhealthy subjects of similar age and sex.

In other embodiments, a therapeutically effective dose or amount of anAAV mini-dystrophin vector of the disclosure is effective to alter thelevels of ALT, AST, LDH, CK and MMP-9 as indicated above alone or incombination with one or more of these same or other biomarkers. Thus, inan exemplary non-limiting embodiment, a therapeutically effective doseor amount of an AAV mini-dystrophin vector of the disclosure iseffective to reduce ALT and AST, ALT and LDH, AST and CK, or AST andMMP-9, etc.

In some methods of treatment of the disclosure, an effective dose oramount of an AAV vector is one that improves average subject performancein the 6 minute walk-test (6MWT). The 6MWT has been established as areproducible and valid measure of muscle function and mobility of humansubjects with muscular dystrophy, in particular, DMD. See, for example,McDonald, C M, et al., Muscle Nerve 41(4):500-10 (2010); Henricson, E,et al., PLOS Currents Musc Dys, 8 Jul. 2013; McDonald, C M, et al.,Muscle Nerve 48:343-56 (2013). In the test, the distance in meters thata subject can, starting from rest, walk continually and unaided during a6 minute period is recorded. This distance is also known as the 6 minutewalk distance (6MWD). In some applications of the test, an individualsubject may be tested more than once over a period of days, and theresults averaged. Due to its advantages, the 6MWT has been adopted as aprimary clinical endpoint in drug trials involving ambulatory DMDpatients. See, for example, Bushby, K, et al., Muscle Nerve 50:477-87(2014); Mendell, J R, et al., Ann Neurol 79:257-71 (2016); Campbell, C,et al., Muscle Nerve 55(4):458-64 (2017). Usually, in these trials, eachsubject in the treatment group has his ambulation tested using the 6MWTover a period of months or years to determine if a treatment effectexists.

According to some embodiments of the methods of treatment of thedisclosure, therapeutic efficacy is determined statistically bycomparing the treatment effect of AAV vectors of the disclosure on theaverage 6MWT performance of treated subjects, such as those with DMD, incomparison with the average 6MWT performance of untreated controlsubjects with the same type of dystrophinopathy, such as DMD. Suchcontrols can have been included in the same studies used to evaluate thetherapeutic efficacy of AAV vectors of the disclosure, or can be similarsubjects drawn from natural history studies of the progression of DMD orother dystrophinopathies. Controls can be age matched (or stratified,for example and without limitation, into those subjects younger than orolder than some threshold age, such as 6, 7, 8, 9, or 10 years), matchedfor status of prior corticosteroid treatment (that is, yes or no, orlength of time of previous treatment), matched for baseline performancein the 6MWT before any treatment (except perhaps with corticosteroids)(or stratified, for example and without limitation, into those subjectswhose baseline performance is below and above some threshold, such as200 m, 250 m, 300 m, 350 m, 400 m, 450 m, or 500 m), or some otherattribute determined to be clinically relevant.

According to certain embodiments of the methods of treatment of thedisclosure, a therapeutically effective dose or amount of an AAV vectorof the disclosure is effective to increase the average 6MWD of subjectswith dystrophinopathy, such as DMD, by about or at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100meters or more compared to similar matched or stratified controls 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, or 36 months after administration ofthe vector. In some of these embodiments, the AAV vector comprises theAAV9 capsid and a genome including a human codon-optimized gene encodinga mini-dystrophin protein, such as, without limitation, the vectordesignated as AAV9.hCK.Hopti-Dys3978.spA.

According to certain embodiments of the methods of treatment of thedisclosure, a therapeutically effective dose or amount of an AAV vectorof the disclosure is effective to increase the average 6MWD of subjectswith dystrophinopathy, such as DMD, by about or at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100meters or more compared to similar matched or stratified controls 30,60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480,510, 540, 570, 600, 630, 660, 690 or 720 days after administration ofthe vector. In some of these embodiments, the AAV vector comprises theAAV9 capsid and a genome including a human codon-optimized gene encodinga mini-dystrophin protein, such as, without limitation, the vectordesignated as AAV9.hCK.Hopti-Dys3978.spA.

As an alternative to the 6MWT, therapeutic efficacy can be expressed asreduction in the time it takes a subject to ascend 4 standard sizedstairs, a test known as the 4 stair climb test. This test has been usedto assess the effectiveness of corticosteroid treatment in DMD patients.Griggs, R C, et al., Arch Neurol 48(4):383-8 (1991). Thus, according tocertain embodiments of the methods of treatment of the disclosure, atherapeutically effective dose or amount of an AAV vector of thedisclosure is effective to reduce the average time it takes for subjectswith dystrophinopathy, such as DMD, to perform the 4 stair climb test byabout or at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2,2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 seconds or more comparedto similar matched or stratified controls 3, 6, 9, 12, 15, 18, 21, 24,27, 30, 33, or 36 months after administration of the vector. In some ofthese embodiments, the AAV vector comprises the AAV9 capsid and a genomeincluding a human codon-optimized gene encoding a mini-dystrophinprotein, such as, without limitation, the vector designated asAAV9.hCK.Hopti-Dys3978.spA.

In related embodiments of the methods of treatment of the disclosure, atherapeutically effective dose or amount of an AAV vector of thedisclosure is effective to reduce the average time it takes for subjectswith dystrophinopathy, such as DMD, to perform the 4 stair climb test byabout or at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2,2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 seconds or more comparedto similar matched or stratified controls 30, 60, 90, 120, 150, 180,210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600,630, 660, 690 or 720 days after administration of the vector. In some ofthese embodiments, the AAV vector comprises the AAV9 capsid and a genomeincluding a human codon-optimized gene encoding a mini-dystrophinprotein, such as, without limitation, the vector designated asAAV9.hCK.Hopti-Dys3978.spA.

Therapeutic efficacy can also be expressed as a reduction over time inthe percentage of subjects that experience loss of ambulation aspecified time after treatment compared to controls. Loss of ambulationis defined as start of continuous reliance on wheelchair use. Thus,according to yet other embodiments of the methods of treatment of thedisclosure, a therapeutically effective dose or amount of an AAV vectorof the disclosure reduces, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, or36 months after administration to subjects with dystrophinopathy, suchas DMD, the average number of subjects that have lost ambulation by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% ormore compared to similar matched or stratified controls. In some ofthese embodiments, the AAV vector comprises the AAV9 capsid and a genomeincluding a human codon-optimized gene encoding a mini-dystrophinprotein, such as, without limitation, the vector designated asAAV9.hCK.Hopti-Dys3978.spA.

In some embodiments of the methods of treatment of the disclosure, atherapeutically effective dose or amount of an AAV vector of thedisclosure is effective to delay the onset of one or more symptoms in asubject having a dystrophinopathy, such as DMD. Diagnosis before onsetof symptoms can be accomplished through prenatal, perinatal or postnatalgenetic testing for mutations in the DMD gene. According to certainembodiments, treatment with an AAV vector of the disclosure is effectiveto delay onset of one or more symptoms of DMD by at least or about 3, 4,5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32,34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 56, 28, 60, 62,64, 65, 66, 68, 70, 72, 74, 75, 76, 78, or 80 months, or more comparedto similar matched or stratified controls. As appreciated by those ofordinary skill, early symptoms of DMD include without limitation delayin walking ability (to an average age of about 18 months, compared to anaverage of 12-15 months in babies without DMD); difficulty jumping,running or climbing stairs; proneness to falling; proximal muscleweakness, evidenced, for example, by exhibiting the Gowers' maneuverwhen rising from the floor; enlarged calves, due to pseudohypertrophy;waddling gait due to subjects' walking on toes and/or balls of feet;tendency to maintain balance by sticking out bellies and pulling backshoulders; and cognitive impairments, such as diminished receptivelanguage, expressive language, visuospatial ability, fine motor skills,attention, and memory skills. In some of these embodiments, the AAVvector comprises the AAV9 capsid and a genome including a humancodon-optimized gene encoding a mini-dystrophin protein, such as,without limitation, the vector designated as AAV9.hCK.Hopti-Dys3978.spA.

Therapeutic efficacy can also be expressed as a reduction over time inthe percentage of vector treated subjects that experience an increase inthe amount of adipose tissue that replaces lean muscle tissue comparedto untreated controls. In some embodiments, this progression towardincreased adiposity can be determined using MRI analysis of the legmuscles of DMD patients and expressed as the fat fraction (FF), asexplained further in Willcocks, R J, et al., Multicenter prospectivelongitudinal study of magnetic resonance biomarkers in a large Duchennemuscular dystrophy cohort, Ann Neurol 79:535-47 (2016). In relatedembodiments, treatment of DMD subjects with an AAV vector of thedisclosure is effective to reduce the average FF in their lowerextremities as determined by MRI by about or at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more 3, 6, 9,12, 15, 18, 21, 24, 27, 30, 33, or 36 months after treatment compared tomatched controls. In some of these embodiments, the AAV vector comprisesthe AAV9 capsid and a genome including a human codon-optimized geneencoding a mini-dystrophin protein, such as, without limitation, thevector designated as AAV9.hCK.Hopti-Dys3978.spA.

In some embodiments, a therapeutically effective dose or amount of anAAV vector of the disclosure is one that results in at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or more of skeletal muscle fibers expressing themini-dystrophin protein 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, or 36months after treatment. The percentage of muscle fibers that arepositive for mini-dystrophin protein expression may be determined byimmunolabeling sections of biopsied muscle from treated subjects with ananti-dystrophin antibody capable of specifically binding themini-dystrophin protein. Suitable immunolabeling techniques aredescribed in the Examples, and are familiar to those of ordinary skillin the art. Exemplary muscles of treated subjects from which biopsiesmay be taken include bicep, deltoid, and quadriceps, although othermuscles may be biopsied as well. In some of these embodiments, the AAVvector comprises the AAV9 capsid and a genome including a humancodon-optimized gene encoding a mini-dystrophin protein, such as,without limitation, the vector designated as AAV9.hCK.Hopti-Dys3978.spA.

In some embodiments, a dose or amount of an AAV vector of the disclosurefor treating dystrophinopathy, such as muscular dystrophy, such as DMD,is determined to be therapeutically effective and at the same timecauses either no cellular (T cell) immune response specific for themini-dystrophin protein in treated subjects, or in only a low percentageof such subjects. Existence or extent of a T cell response against themini-dystrophin protein can be determined using the ELISPOT assay todetect peripheral blood mononuclear cells (PBMCs) isolated from subjectblood that produce gamma interferon (IFNγ) in response to exposure to anoverlapping peptide library covering the mini-dystrophin protein aminoacid sequence. In certain embodiments, the threshold for a positive IFNγresponse can be set as greater than 50 spot-forming cells per millionPBMCs tested. Use of other assays to detect a T cell response againstthe mini-dystrophin protein are also possible including withoutlimitation detection of T cell infiltrates in biopsies of muscle orother tissues expressing mini-dystrophin protein obtained from vectortreated subjects. Subjects can be human subjects or animal subjects,such as animal models of DMD, such as the mdx mouse, mdx rat, or GRMDdog models. In other embodiments, a dose or amount of an AAV vector ofthe disclosure for treating dystrophinopathy, such as musculardystrophy, such as DMD, is determined to be therapeutically effectiveand at the same time causes either no inflammatory response against thecapsid, vector genome (or any component thereof), or mini-dystrophinprotein expressed by transduced cells, or in only a low percentage ofsuch subjects. Without wishing to be bound by any particular theory ofoperation, inflammation in response to an AAV vector may be caused by aninnate immune response. Inflammation, if any exists, in the muscles ofvector treated subjects can be detected using magnetic resonanceimaging. See, for example, J Garcia, Skeletal Radiol 29:425-38 (2000)and Schulze, M, et al., Am J Radiol 192:1708-16 (2009). Subjects can behuman subjects or animal subjects, such as animal models of DMD, such asthe mdx mouse, mdx rat, or GRMD dog models. In some of the embodimentsdescribed above, existence or absence of cellular immune response orinflammation is determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36 months after treatment, or some other time aftertreatment. In related embodiments, a low percentage of subjectsexhibiting a cellular immune response to the mini-dystrophin proteinwould be less than or equal to about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of subjectsadministered vector. In some of these embodiments, the AAV vectorcomprises the AAV9 capsid and a genome including a human codon-optimizedgene encoding a mini-dystrophin protein, such as, without limitation,the vector designated as AAV9.hCK.Hopti-Dys3978.spA.

In related embodiments, a dose or amount of an AAV vector of thedisclosure for treating dystrophinopathy, such as muscular dystrophy,such as DMD, is therapeutically effective without need for concomitantimmune suppression in treated subjects. Thus, in certain embodiments,treatment of a subject with dystrophinopathy, such as DMD, is effectivewithout need to administer to the subject before, during or aftertreatment with AAV vector one or more immune-suppressing drugs (apartfrom steroid treatment, which is the current standard of care).Exemplary immune-suppressing drugs include but are not limited tocalcineurin inhibitors, such as tacrolimus and cyclosporin,antiproliferative agents, such as mycophenolate, leflunomide, andazathioprine, or mTOR inhibitors, such as sirolimus and everolimus.

As explored in greater detail in the Examples, efficacy of the AAVvectors of the disclosure, including without limitation the vectordesignated as AAV9.hCK.Hopti-Dys3978.spA, can be tested in animal modelsof Duchenne muscular dystrophy, and results used to predict efficaciousdoses of such vectors in human DMD patients. Various animal models areknown in the art, including the mdx mouse model, the Golden Retrievermuscular dystrophy model, and more recently, the Dmd^(mdx) rat model,which is described in greater detail in the Examples.

Based on the Dmd^(mdx) rat model, effective doses of AAV vectors of thedisclosure, including the vector designated asAAV9.hCK.Hopti-Dys3978.spA, can be established with respect to variousbiological parameters and aspects of the disease course in the rats.

Thus, according to certain embodiments of the disclosure, treatment ofDmd^(mdx) rats with a dose of AAV9.hCK.Hopti-Dys3978.spA of at least1×10¹⁴ vg/kg or 3×10¹⁴ vg/kg is effective to reduce serum AST, ALT, LDH,or total creatine kinase levels at 3 months or 6 months post-injectioncompared to controls.

In other embodiments, treatment of Dmd^(mdx) rats with a dose ofAAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kg iseffective to reduce fibrosis in biceps femoris, diaphragm, or heartmuscle at 3 months or 6 months post-injection compared to controls.

In yet other embodiments, treatment of Dmd^(mdx) rats with a dose ofAAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kg iseffective to increase forelimb grip force at 3 months or 6 monthspost-injection compared to controls.

According to other embodiments, treatment of Dmd^(mdx) rats with a doseof AAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kgis effective to reduce muscle fatigue as measured over 5 closely spacedtrials testing forelimb grip force at 3 months or 6 monthspost-injection compared to controls.

In some other embodiments, treatment of Dmd^(mdx) rats with a dose ofAAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kg iseffective to increase the left ventricular ejection fraction as measuredusing echocardiography at 6 months post-injection compared to controls.

In other embodiments, treatment of Dmd^(mdx) rats with a dose ofAAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kg iseffective to increase the ratio of the velocity of early to late leftventricular filling (i.e., E/A ratio) as measured using echocardiographyat 3 months or 6 months post-injection compared to controls.

According to some embodiments, treatment of Dmd^(mdx) rats with a doseof AAV9.hCK.Hopti-Dys3978.spA of at least 1×10¹⁴ vg/kg or 3×10¹⁴ vg/kgis effective to decrease the isovolumetric relaxation time (IVRT) or thetime in milliseconds between peak E velocity and its return to baseline(i.e., the E wave deceleration time (DT)) as measured usingechocardiography at 3 months or 6 months post-injection compared tocontrols.

In each of the foregoing embodiments, the increase or decrease of thephysiologic measurement in vector-treated animals compared to controlanimals can, in some embodiments, be tested for statisticalsignificance. The choice of which statistical test to apply is withinthe knowledge of those ordinarily skilled in the art. Where a p-value isadopted as the way in which to assess statistical significance, suchp-values, once calculated, can be compared to a predefined significancelevel, and if the p-value is smaller than the significance level, thetreatment effect can be determined to be statistically significant. Insome embodiments, the significance level can be predefined as 0.25,0.20, 0.15, 0.10, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or some othersignificance level. Thus, in an exemplary non-limiting embodiment, wherethe significance level is predefined as 0.05, then calculation of ap-value<0.05 would be interpreted to represent a statisticallysignificant difference between vector-treated and control groups.

In each of the foregoing embodiments, the controls can be age matchedanimals of the same sex and genetic background that are untreated, ortreated only with vehicle and not vector. Other controls are alsopossible, however.

In some other embodiments, treatment of Dmd^(mdx) rats with a dose ofAAV9.hCK.Hopti-Dys3978.spA of at least 3×10¹⁴ vg/kg is effective totransduce biceps femoris, diaphragm, heart muscle, or other striatedmuscles, and express the mini-dystrophin protein encoded by theopti-Dys3978 gene without inducing a cellular immune response againstthe mini-dystrophin protein by 3 months or 6 months post-injection.Cellular immune response against the mini-dystrophin protein can beassessed by isolating splenocytes, or blood lymphocytes, such asperipheral blood mononuclear cells (PBMCs), from test animals,incubating the cells with peptides from an overlapping peptide librarycovering the mini-dystrophin protein amino acid sequence (for example,peptides 15 amino acids long overlapping by 10 amino acids each) inpools (for example, 5 pools), and determining whether the cells producegamma interferon (IFNγ) in response to being exposed to the peptides.Production of IFNγ can be determined using the ELISPOT assay accordingto the knowledge of those ordinarily skilled in the art. See, forexample, Smith, J G, et al., Clin Vaccine Immunol 8(5):871-9 (2001),Schmittel A, et al., J Immunol Methods 247:17-24 (2001), and Marino, AT, et al., Measuring immune responses to recombinant AAV gene transfer,Ch. 11, pp. 259-72, Adeno-Associated Virus Methods and Protocols, Ed. RO Snyder and P Moullier, Humana Press (2011). In certain embodiments,the threshold for a positive IFNγ response can be set as greater than 50spot-forming cells per million cells tested, or in other embodiments, asat least 3-times the number of spot-forming cells detected using anegative control (for example, medium only without added peptides), sothat a negative response would be considered below these thresholds.

In some embodiments of the methods of treatment of the presentdisclosure, an AAV vector for treating dystrophinopathy, such as DMD, isadministered to a subject in need of treatment for dystrophinopathy,such as DMD, jointly with at least a second agent established orbelieved to be effective for treating dystrophinopathy, such as DMD.Joint administration of the AAV vector means treating a subject before,contemporaneously with, or after treatment of the second agent.According to certain embodiments, the AAV vector is jointly administeredwith an antisense oligonucleotide that causes exon skipping of the DMDgene, for example of exon 51 of the dystrophin gene, or some other exonof the dystrophin gene. Agents that cause skipping of exon 51 of thedystrophin gene include drisapersen and eteplirsen, but others arepossible. In other embodiments, the AAV vector is jointly administeredwith an agent that inhibits myostatin function in the subject, such asan anti-myostatin antibody, examples of which are provided in U.S. Pat.Nos. 7,888,486, 8,992,913, and 8,415,459. In other embodiments, wherethe dystrophinopathy of the subject can be attributed to a nonsensemutation in the dystrophin gene, the AAV vector is jointly administeredwith an agent that promote ribosomal read-through of nonsense mutations,such as ataluren, or with an agent that suppresses premature stopcodons, such as an aminoglycoside, such as gentamicin. In otherembodiments, the AAV vector is jointly administered with an anabolicsteroid, such as oxandrolone. And in yet other embodiments, the AAVvector is jointly administered with a corticosteroid, such as withoutlimitation prednisone, deflazacort, or prednisolone. In some embodimentsof these methods, the AAV vector is an AAV9 vector comprising a genomeincluding a human codon-optimized gene encoding a mini-dystrophinprotein, such as, without limitation, the vector designated asAAV9.hCK.Hopti-Dys3978.spA.

Pharmaceutical Formulations and Modes of Administration

Virus vectors and capsids according to the present invention find use inboth veterinary and human medical applications. Suitable subjectsinclude both avians and mammals. The term “avian” as used hereinincludes, but is not limited to, chickens, ducks, geese, quail, turkeys,pheasant, parrots, parakeets, and the like. The term “mammal” as usedherein includes, but is not limited to, humans, non-human primates,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects include neonates, infants, juveniles and adults.Optionally, the subject is “in need of” the methods of the presentinvention, e.g., because the subject has or is believed at risk for adisorder including those described herein or that would benefit from thedelivery of a polynucleotide including those described herein. As afurther option, the subject can be a laboratory animal and/or an animalmodel of disease.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector (such as an rAAVparticle) and/or capsid of the invention in a pharmaceuticallyacceptable carrier and, optionally, other medicinal agents,pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants,diluents, etc. For injection, the carrier will typically be a liquid.For other methods of administration, the carrier may be either solid orliquid. For inhalation administration, the carrier will be respirable,and optionally can be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring apolynucleotide encoding mini-dystrophin to a cell in vitro. The virusvector may be introduced into the cells at the appropriate multiplicityof infection according to standard transduction methods suitable for theparticular target cells. Titers of virus vector to administer can vary,depending upon the target cell type and number, and the particular virusvector, and can be determined by those of skill in the art without undueexperimentation. In representative embodiments, at least about 10³infectious units, more preferably at least about 105 infectious unitsare introduced to the cell.

The cell(s) into which the virus vector is introduced can be of anytype, including but not limited to muscle cells (e.g., skeletal musclecells, cardiac muscle cells, smooth muscle cells and/or diaphragm musclecells), stem cells, germ cells, and the like. In representativeembodiments, the cell can be any progenitor cell. As a furtherpossibility, the cell can be a stem cell (e.g., muscle stem cell).Moreover, the cell can be from any species of origin, as indicatedabove.

The virus vector can be introduced into cells in vitro for the purposeof administering the modified cell to a subject. In particularembodiments, the cells have been removed from a subject, the virusvector is introduced therein, and the cells are then administered backinto the subject. Methods of removing cells from subject formanipulation ex vivo, followed by introduction back into the subject areknown in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively,the recombinant virus vector can be introduced into cells from a donorsubject, into cultured cells, or into cells from any other suitablesource, and the cells are administered to a subject in need thereof(i.e., a “recipient” subject).

Suitable cells for ex vivo gene delivery are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ cells or at least about 10³to about 10⁶ cells will be administered per dose in a pharmaceuticallyacceptable carrier. In particular embodiments, the cells transduced withthe virus vector are administered to the subject in a treatmenteffective or prevention effective amount in combination with apharmaceutical carrier.

A further aspect of the invention is a method of administering the virusvector to subjects. Administration of the virus vectors and/or capsidsaccording to the present invention to a human subject or an animal inneed thereof can be by any means known in the art. Optionally, the virusvector and/or capsid is delivered in a treatment effective or preventioneffective dose in a pharmaceutically acceptable carrier.

Dosages of the virus vector and/or capsid to be administered to asubject depend upon the mode of administration, the disease or conditionto be treated and/or prevented, the individual subject's condition, theparticular virus vector or capsid, and the nucleic acid to be delivered,and the like, and can be determined in a routine manner. Exemplary dosesfor achieving therapeutic effects are titers of at least about 10⁵, 10⁶, 10⁷, 10 ⁸, 10⁹, 10 ¹⁰, 10¹¹, 10 ¹², 10¹³, 10¹⁴, 10¹⁵ transducingunits, optionally about 10⁸-10¹³ transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

In certain embodiments, an AAV vector or particle of the disclosure canbe administered to a subject in compositions comprising empty AAVcapsids of the same or a different serotype. Empty capsids are AAVcapsids comprising the typical arrangement and ratios of VP1, VP2 andVP3 capsid proteins, but do not contain a vector genome. Without wishingto be bound by any particular theory of operation, it is hypothesizedthat the presence of empty capsids can reduce the immune responseagainst the capsid of the AAV vector, and thereby increase transductionefficiency. Empty capsids can occur naturally in a preparation of AAVvector, or be added in known quantities to achieve known ratios of emptycapsids to AAV vector (that is, capsids containing vector genomes).Preparation, purification and quantitation of empty capsids is withinthe knowledge of those ordinarily skilled in the art. Compositionscomprising AAV vectors of the disclosure and empty capsids can beformulated with an excess of empty capsids relative to AAV vectors, oran excess of genome containing AAV vectors relative to empty capsids.Thus, in some embodiments, compositions of the disclosure comprise AAVvectors of the disclosure and empty capsids of the same or a differentserotype, wherein the ratio of empty capsids to AAV vectors is about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10 to 1, or some other ratio.

In other embodiments, the disclosure provides exemplary efficaciousdoses of AAV vector particles for treating dystrophinopathy, such asmuscular dystrophy, such as DMD, quantified as vector genomes (vg) perkilogram of subject body weight (kg), abbreviated vg/kg. According tocertain embodiments, an efficacious dose of an AAV vector of thedisclosure, including those comprising an AAV9 capsid and a genomeincluding a human codon-optimized gene encoding a mini-dystrophinprotein, such as, without limitation, the vector designated asAAV9.hCK.Hopti-Dys3978.spA, is about 1×10¹² vg/kg, 2×10¹² vg/kg, 3×10¹²vg/kg, 4×10¹² vg/kg, 5×10¹² vg/kg, 6×10¹² vg/kg, 7×10¹² vg/kg, 8×10¹²vg/kg, 9×10¹² vg/kg, 1×10¹³ vg/kg, 2×10¹³ vg/kg, 3×10¹³ vg/kg, 4×10¹³vg/kg, 5×10¹³ vg/kg, 6×10¹³ vg/kg, 7×10¹³ vg/kg, 8×10¹³ vg/kg, 9×10¹³vg/kg, 1×10¹⁴ vg/kg, 1.5×10¹⁴ vg/kg, 2×10¹⁴ vg/kg, 2.5×10¹⁴ vg/kg,3×10¹⁴ vg/kg, 3.5×10¹⁴ vg/kg, 4×10¹⁴ vg/kg, 5×10¹⁴ vg/kg, 6×10¹⁴ vg/kg,7×10¹⁴ vg/kg, 8×10¹⁴ vg/kg, or 9×10¹⁴ vg/kg, or some other dose. In anyof these embodiments, the AAV vector may be administered to a subject ina pharmaceutically acceptable composition alone, or with empty capsidsof the same capsid serotype at an empty capsid to vector ratio of about0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or some otherratio.

Exemplary modes of administration include oral, rectal, transmucosal,intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal,intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous,subcutaneous, intradermal, intracranial, intramuscular (includingadministration to skeletal, diaphragm and/or cardiac muscle),intrapleural, intracerebral, and intra-articular), topical (e.g., toboth skin and mucosal surfaces, including airway surfaces, andtransdermal administration), intra-lymphatic, and the like, as well asdirect tissue or organ injection (e.g., to skeletal muscle, cardiacmuscle, or diaphragm muscle).

Administration can be to any site in a subject, including, withoutlimitation, a site selected from the group consisting of a skeletalmuscle, a smooth muscle, the heart, and the diaphragm.

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscle in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscles include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii superioris, levatorlabii superioris alaeque nasi, levator palpebrae superioris, levatorscapulae, long rotators, longissimus capitis, longissimus cervicis,longissimus thoracis, longus capitis, longus colli, lumbricals (in thehand), lumbricals (in the foot), masseter, medial pterygoid, medialrectus, middle scalene, multifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor,and any other suitable skeletal muscle as known in the art.

The virus vector can be delivered to skeletal muscle by intravenousadministration, intra-arterial administration, intraperitonealadministration, limb perfusion, (optionally, isolated limb perfusion ofa leg and/or arm; see, e.g. Arruda et al., (2005) Blood 10⁵: 3458-3464),and/or direct intramuscular injection. In particular embodiments, thevirus vector and/or capsid is administered to a limb (arm and/or leg) ofa subject (e.g., a subject with muscular dystrophy such as DMD) by limbperfusion, optionally isolated limb perfusion (e.g., by intravenous orintra-articular administration. In embodiments of the invention, thevirus vectors and/or capsids of the invention can advantageously beadministered without employing “hydrodynamic” techniques. Tissuedelivery (e.g., to muscle) of prior art vectors is often enhanced byhydrodynamic techniques (e.g., intravenous/intravenous administration ina large volume), which increase pressure in the vasculature andfacilitate the ability of the vector to cross the endothelial cellbarrier. In particular embodiments, the viral vectors and/or capsids ofthe invention can be administered in the absence of hydrodynamictechniques such as high volume infusions and/or elevated intravascularpressure (e.g., greater than normal systolic pressure, for example, lessthan or equal to a 5%, 10%, 15%, 20%, 25% increase in intravascularpressure over normal systolic pressure). Such methods may reduce oravoid the side effects associated with hydrodynamic techniques such asedema, nerve damage and/or compartment syndrome.

Administration to cardiac muscle includes administration to the leftatrium, right atrium, left ventricle, right ventricle and/or septum. Thevirus vector and/or capsid can be delivered to cardiac muscle byintravenous administration, intra-arterial administration such asintra-aortic administration, direct cardiac injection (e.g., into leftatrium, right atrium, left ventricle, right ventricle), and/or coronaryartery perfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Administration to smooth muscle can be by any suitable method includingintravenous administration, intra-arterial administration, and/orintra-peritoneal administration. In one embodiment, administration canbe to endothelial cells present in, near, and/or on smooth muscle.

Delivery to a target tissue can also be achieved by delivering a depotcomprising the virus vector and/or capsid. In representativeembodiments, a depot comprising the virus vector and/or capsid isimplanted into skeletal, smooth, cardiac and/or diaphragm muscle tissueor the tissue can be contacted with a film or other matrix comprisingthe virus vector and/or capsid. Such implantable matrices or substratesare described in U.S. Pat. No. 7,201,898.

In particular embodiments, a virus vector according to the presentinvention is administered to skeletal muscle, diaphragm muscle and/orcardiac muscle (e.g., to treat and/or prevent muscular dystrophy).

In representative embodiments, the invention is used to treat and/orprevent disorders of skeletal, cardiac and/or diaphragm muscle.

In a representative embodiment, the invention provides a method oftreating and/or preventing muscular dystrophy in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding dystrophin, a mini-dystrophin, or a micro-dystrophin. Inparticular embodiments, the virus vector can be administered toskeletal, diaphragm and/or cardiac muscle as described elsewhere herein.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector and/or virus capsids of theinvention in a local rather than systemic manner, for example, in adepot or sustained-release formulation. Further, the virus vector and/orvirus capsid can be delivered adhered to a surgically implantable matrix(e.g., as described in U.S. Patent Publication No. 2004-0013645).

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Synthesis of Codon-Optimized Human Mini-Dystrophin Genes

Previously we generated a number of miniature versions of humandystrophin gene by PCR cloning of human muscle dystrophin cDNA,generating mini-dystrophin genes that have large deletions in thecentral rod domain and nearly complete deletion of the C-terminal regionof the dystrophin coding sequence (Wang et al., Proc. Natl. Acad. Sci.,USA 97:13714 (2000); U.S. Pat. Nos. 7,001,761 and 7,510,867). Thesemini-dystrophin genes were tested to be highly functional in vivo in DMDmdx mouse models (Watchko et al., Human Gene Therapy 13:1451 (2002)).One of these mini-dystrophin proteins, named Δ3990, was described inU.S. Pat. No. 7,510,867 under SEQ ID NO:6. The protein sequence of Δ3990and the DNA encoding it are provided herein by SEQ ID NO:27 and SEQ IDNO:28, respectively.

A modification of the Δ3990 mini-dystrophin was also designed,codon-optimized, and tested for activity. This new humanmini-dystrophin, called Dys3978, is 1325 amino acids in length, andincludes the following portions or subdomains from wildtype full-lengthhuman muscle dystrophin (SEQ ID NO:25): the N-terminus and actin-bindingdomain (ABD), hinge H1, rods R1 and R2, hinge H3, rods R22, R23 and R24,hinge H4, the cysteine-rich domain (CR domain) and part of thecarboxy-terminal domain (CT domain). The amino acid sequence of thisprotein is provided by SEQ ID NO:7 and is illustrated schematically inFIG. 1 . To reduce potential immunogenicity, the last four amino acidsat the C-terminus of the Δ3990 protein were deleted. In creating Δ3990,this sequence had been formed by joining part of the amino-terminal endof the dystrophin carboxy-terminal domain (ending at P3409) with thelast three amino acids of dystrophin (3683-3685, or DTM). This stretchof four amino acids has no known function and could function as a newepitope because the sequence does not occur in wildtype dystrophin. Inaddition, a valine at amino acid position 2 in Δ3990, not present inwildtype dystrophin, but which resulted from creation of a consensusKozak initiation sequence around the start codon of Δ3990 was changed tothe leucine ordinarily present in dystrophin. Thus there are 5 aminoacid differences between Δ3990 and Dys3978. An amino acid sequencealignment between Δ3990 and Dys3978 is provided in FIGS. 55A-55C.

The gene encoding Dys3978 was constructed by combining subsequences fromthe wildtype dystrophin coding sequence corresponding to the proteinsubdomains described above. The resulting gene is provided by SEQ IDNO:26. To increase the expression of Dys3978, the gene sequence wascodon-optimized using human codon algorithms. The resulting humancodon-optimized gene, called Hopti-Dys3978, is provided as SEQ ID NO:1.A canine codon-optimized gene encoding Dys3978, called Copti-Dys3978,was also generated, the sequence of which is provided as SEQ ID NO:3. Analignment comparing the DNA sequences of Hopti-Dys3978 and thenon-codon-optimized gene encoding Δ3990 is provided in FIGS. 56A-56I.

Among other changes, codon-optimization of the gene encoding Dys3978increased total GC content from about 46% in the non-codon-optimizedgene to about 61% in the human codon-optimized gene (i.e.,Hopti-Dys3978). Increasing GC content can result in increased mRNAlevels in mammalian cells. See, for example, Grzegorz, K, et al., PLoSBiol, 4(6):e180 (2006); and Newman, Z R, et al., PNAS, E1362-71 (2016).Codon-optimization also increased the codon adaptation index (CAI) andincluded addition of a Kozak consensus transcription initiationrecognition site at the beginning of the coding sequence.

To examine if human codon optimization could enhance gene expression,the Hopti-Dys3978 gene was cloned into an AAV vector expression cassettecontaining the constitutively active CMV promoter and a small syntheticpolyadenylation (polyA) signal sequence (SEQ ID NO: 6). Aftertransfection into human HEK 293 cells, the vector plasmid containing theHopti-Dys3978 gene showed surprisingly greater protein expression thanthe non-optimized gene encoding Dys3978, as determined qualitativelyusing immunofluorescent staining and Western blot against dystrophinprotein (FIG. 2 ).

A gene encoding a human mini-dystrophin similar in structure to Dys3978,except that hinge H3 is absent, was also generated and codon-optimized.This gene, called Hopti-Dys3837 (SEQ ID NO: 2) encodes a humanmini-dystrophin protein of 1278 amino acids called Dys3837 (SEQ ID NO:8), which is also illustrated schematically in

FIG. 1 .

For other experiments described herein, the human and caninecodon-optimized Dys3978 genes were placed under the control of one oftwo different synthetic muscle-specific promoter and enhancercombinations derived from the muscle creatine kinase gene identifiedbelow:

-   -   1) Synthetic hybrid muscle-specific promoter (hCK) (SEQ ID NO:        4); and    -   2) Synthetic hybrid muscle-specific promoter plus (hCKplus) (SEQ        ID NO: 5);

For use in the experiments, the following vectors were constructed usingstandard molecular cloning techniques. The gene expression cassettes ofthe specified promoter, mini-dystrophin gene and polyA sequence werecloned into an AAV vector plasmid backbone containing AAV2 invertedterminal repeats (ITRs) flanking the expression cassette.

1) AAV-CMV-Hopti-Dys3978 (SEQ ID NO: 9) 2) AAV-hCK-Hopti-Dys3978(SEQ ID NO: 10) 3) AAV-hCK-Hopti-Dys3837 (SEQ ID NO: 11)4) AAV-hCKplus-Hopti-Dys3837 (SEQ ID NO: 12) 5) AAV-hCK-Copti-Dys3978

Example 2 CMV-Hopti-Dys3978 in Dystrophin/Utrophin Double Knockout Mice

The loss of dystrophin in the patients of Duchenne muscular dystrophy(DMD) results in devastating skeletal muscle degeneration andcardiomyopathy. Mdx mice lacking only dystrophin have a much milderphenotype, whereas double knockout (dKO) mice lacking both dystrophinand its homolog utrophin exhibit the similarly severe dystrophicclinical signs seen in DMD patients. It was previously demonstrated thatintraperitoneal injection in neonatal homozygous dKO mice with 3×10¹¹vg/mouse of AAV1-CMV-Δ3990 (not codon-optimized) was able to partiallyrestore growth, functions and prolong life-span for a few months (50%survival rate at 22 weeks) (see FIG. 6B from Wang et al., J. Orthop.Res, 27:421 (2009)). Here, the therapeutic effects of systemic deliveryof human codon-optimized Hopti-Dys3978 gene were evaluated using AAV9 asthe capsid. The results demonstrate that a single systemicadministration (IP) of AAV9-CMV-Hopti-Dys3978 at about 2×10¹³ vg/kg into1-week-old neonatal dKO mice led to widespread expression of themini-dystrophin gene in skeletal muscles and in the entire heart muscle(FIG. 3 ). The AAV9-treated dKO mice showed near normal growth curve andbody weight (FIG. 4 ) and significantly improved muscle function asevaluated by the grip force and treadmill running tests (FIG. 5 ). Thetreated dKO mice also showed amelioration of dystrophic pathology (FIGS.6A-6B) and great improvement of overall health. When compared to the dKOmice treated with an AAV1 vector expressing non-codon-optimized Δ3990,the dKO mice treated with Hopti-Dys3978 gene showed a much prolongedlife-span (50% survival rate: 22 weeks vs. more than 80 weeks) (FIG. 7). Unexpectedly, the fertility of both male and female dKO mice wererestored (Table 1), suggesting overall function improvement and possiblyimprovement in smooth muscle function as well.

TABLE 1 Mini-dystrophin restores fertility of dKO mice Breeding pairs:Pair #1: T-dKO male X T-dKO female 5 pups Pair #2: T-dKO male X T-dKOfemale 4 pups Pair #3: T-dKO male X T-dKO female 0 pups Pair #4: mdxmale X T-dKO female 5 pups Pair #5: mdx male X T-dKO female 6 pups

The untreated dKO mice are completely infertile. However fertility wasrestored by AAV-CMV-Hopti-Dys3978 in both males and females of treateddKO (T-dKO) mice.

The results described above demonstrate that systemic delivery ofcodon-optimized Hopti-Dys3978 gene was more efficacious than thenon-codon optimized Δ3990 gene.

Importantly, great improvement in cardiac functions was also observed.Therapeutic effects in the heart were evaluated at 4 months of age byhemodynamic analysis using the Millar Pressure-volume system. UntreateddKO mice barely survived over 4 months. The very small body size,kyphosis and severe muscle and cardiac dysfunctions made dKO mice toosick to tolerate the hemodynamic analysis procedure. Therefore, theAAV9-treated dKO mice were compared with untreated, age-matched mdx micewhich had much milder phenotypes due to an intact utrophin gene, whichis known to compensate for lack of dystrophin in this model. Whilemeasurement by echocardiography showed mdx mice had no apparent cardiacdeficit under baseline condition when compared with C57/B10 wildtypemice, they did show apparent deficits as measured by hemodynamics at thebaseline (FIG. 8 , open bars). The results herein show that theAAV9-treated dKO mice displayed similar baseline cardiac hemodynamics tothat of the mdx mice, including end-systolic pressure, end-diastolicvolume, maximal rate of isovolumic contraction (dp/dt_(max)) and maximalrate of isovolumic relaxation (dp/dt min). However, after challenge withdobutamine, treated dKO mice displayed similar baseline cardiachemodynamics to that of the mdx mice, including end-systolic pressure,end-diastolic volume, maximal rate of isovolumic contraction(dp/dt_(max) and dp/dt_(min)), whereas the AAV9-treated dKO miceperformed significantly better than mdx mice in every parameter examined(FIG. 8 , filled bars). Furthermore, greater than 50% of the mdx micedied within the 30-min dobutamine challenge window, consistent with ourprevious report (Wu et al., Proc. Natl. Acad. Sci. USA 10⁵:14814(2008)). In striking contrast, due to cardiac expression of themini-dystrophin transgene in the AAV9-treated dKO mice,dobutamine-induced heart failure was largely prevented. Greater than 90%of the AAV9-treated dKO mice survived the dobutamine stress test in the30 min window. Finally, the commonly seen PR interval deficit shown inelectrocardiograms (ECG) was also improved (FIGS. 9A-9B). The PRinterval is time from the onset of the P wave to the start of the QRScomplex. Taken together, these results demonstrate the effectiveness ofAAV9-CMV-Hopti-Dys3978 gene therapy for cardiomyopathy in a severe DMDmouse model.

Example 3 hCK-Hopti-Dys3978 in Mdx Mice

To examine if the hybrid synthetic muscle-specific promoter hCK was ableto effectively drive Dys3978 gene expression, it was compared with thesame construct driven by the strong non-specific CMV promoter.Immunofluorescent staining of mini-dystrophin expression in mdx micefollowing tail vein injection of the respective vectors showed that thetwo promoters, i.e., hCK and CMV, delivered equivalent expression levelsin muscle and heart (FIG. 10 ).

Example 4 CMV-Hopti-Dys3978 in DMD Canine Model Gold Retriever MuscularDystrophy (GRMD) Dogs

Based on studies in the mdx mice and dystrophin/utrophin double KO (dKO)mice, the same vector, AAV9-CMV-Hopti-Dys3978 was tested in the goldenretriever muscular dystrophy (GRMD) dog, a large animal DMD model.Specifically, the vector was administered to a 2.5-month-old GRMD dog,“Jelly,” and then followed for more than 8 years post injection.

Experimental procedures: GRMD dog “Jelly” (2.5 months old female; 6.3kg; serum CK: 20262 units/L before treatment) was injected withAAV9-CMV-Hopti-Dys3978 vector at a dose of 1×10¹³ vg/kg via the righthind limb. Under general anesthesia, a rubber tourniquet was positionedat the proximal pelvic extremity (the groin area) to cover a majority ofmuscles in the right hind limb. The AAV9 vector was injected via thegreat saphenous vein using a Harvard pump set at injection speed 1ml/sec. The vector volume was 20 ml/kg body weight (130 ml total). Thetourniquet was released after 10 minutes accounting from the start ofinjection. Muscles in the injected limb became harder as revealed bypalpation. MRI images on the hind limbs were collected at about 1 hourpost injection and confirmed vector fluid in the injected limb (FIG. 11). No immuno-suppressant such as steroid was used at any time pointthroughout the more than 8 years of observation. Muscle biopsyprocedures were performed at 5 time points up to 4 years post vectorinjection. Final necropsy was done at the age of 8 years, 4 months, atwhich time “Jelly” was still ambulant but much less active than before.

Results: Immunofluorescent (IF) staining showed long-termmini-dystrophin expression in a majority of muscle samples examined upto final necropsy. Interestingly, the injected limb initially (at 2months post-injection biopsy) had lower expression than the non-injectedlimb, suggesting procedure-related inflammation and partial inactivationof the CMV promoter (FIG. 12 ). Nonetheless, the human mini-dystrophinexpression persisted for 8 years in “Jelly” despite initial inflammationin the injected limb. Muscle biopsies and immunofluorescent staining andWestern blot of the human mini-dystrophin at subsequent time points (7months, 1 year, 2 years, and 4 years post vector injection) showedpersistent gene expression (FIGS. 13-17 ). While the percentages ofmini-dystrophin-positive myofibers varied among different muscles,certain muscles had greater than 90% of myofibers positive upon necropsy(FIG. 18 ). Co-staining of mini-dystrophin and revertant myofibers(anti-C-terminus antibody) showed co-existence of both (FIG. 19 ).Mini-dystrophin was also observed in approximately 20% of thecardiomyocytes (FIG. 18 ). Overall gene expression was largely stable.For example, positive myofibers in the cranial sartorius muscle remainedcomparable throughout the 6 time points, from 2 and 7 months to 1, 4 and8 years (compare FIGS. 12, 13, 14, 17 and 18 ). Western blot confirmedthe IF staining results (FIG. 20 ).

Contractile force measurement showed partial improvement when comparedto the untreated dogs (FIG. 21 ). “Jelly” remained ambulant throughoutthe greater than 8 year post treatment period of observation and waseuthanized due to cardiomyopathy in the final year. No tumors were foundin any of the tissues upon necropsy and examination by a pathologist.DNA sequencing showed that “Jelly” did not carry the disease-modifyingJagged 1 mutation found in two phenotypically mild GRMD dogs as recentlyreported (Vieira et al., Cell 163:1204 (2015)).

Example 5 hCK-Copti-Dys3978 in GRMD Dog

In this study, AAV9-hCK-Copti-Dys3978 vector (a modified creatine kinasepromoter driving a canine codon-optimized human mini-dystrophin 3978)was used in a GRMD dog named “Dunkin.” The gene encodes the same humanmini-dystrophin Dys3978 protein used in other studies, but was caninecodon-optimized. The DNA sequence is 94% identical to the humancodon-optimized gene. Transfection experiments in human HEK 293 cellscomparing CK-Copti-Dys3978 (canine codon-optimized) and CK-Hopti-Dys3978(human codon-optimized) revealed essentially the same level ofexpression. Multiple experiments comparing both constructs in mdx micealso showed essentially the same expression levels.

Experimental procedure: GRMD dog “Dunkin” (female, 2.5 m old, 6.5 kg)was intravenously injected with AAV9-hCK-Copti-Dys3978 vector at thedose of 4×10¹³ vg/kg via the great saphenous vein. The dog was notsedated during injection. There was no noticeable adverse reaction orbehavior change. A muscle biopsy was done 4 months post vector injectionand necropsy was done at 14 months post injection.

Results: Very high level and nearly uniform mini-dystrophin expressionwas observed by immunofluorescent staining of mini-dystrophin 3978 onskeletal muscle samples from 4-month post injection biopsy (FIG. 22 ) to14-month post injection necropsy (FIGS. 23-26 for necropsy).

Significantly high levels of mini-dystrophin in cardiac muscles was alsoobserved by IF staining (FIG. 27 ). The expression from the CK promoterappeared stronger and more uniform than from the CMV promoter.

Western blot analysis confirmed the IF staining results. In the skeletalmuscles, the mini-dystrophin levels were mostly higher than the normallevel of wildtype dystrophin from the normal dog control (FIG. 28 ). Thelevel of Dys3978 in the heart was roughly half that of the wildtypedystrophin level (FIG. 29 ).

Expression of Dys3978 from the canine codon-optimized gene Copti-Dys3978effectively restored dystrophin associated protein complex includinggamma-sarcoglycan (FIG. 30 ).

Quantitative PCR of vector DNA copy numbers showed a consistent trend tothe mini-dystrophin protein expression levels (FIG. 31 ).

There was no innate or cellular immune responses found in all thesamples examined. This is very different from the results ofAAV9-CMV-opH-dys3978, suggesting the muscle-specific hCK promoter wasnot only strong but also safer than the CMV promoter.

Dystrophic pathology was largely ameliorated as shown by H&E stainingfor histology of the heart (FIG. 32 ), diaphragm (FIG. 33 ) and limbmuscles (FIG. 34 ). Trichrome Mason blue staining also showedsignificant reduction of fibrosis in limb muscle and diaphragm (FIG. 35).

Example 6 Preparation of AAV9.hCK.Hopti-Dys3978.spA Vector for In VivoExperiments

The AAV9.hCK.Hopti-Dys3978.spA vector used in Dmd^(mdx) rat studiesdescribed further in Examples 7, 8 and 9 includes an AAV9 capsid and anexpression cassette designed to express a miniaturized version of humandystrophin protein including the N-terminus region, hinge 1 (H1), rod 1(R1), rod 2 (R2), hinge 3 (H3), rod 22 (R22), rod 23 (R23), rod 24(R24), hinge 4 (H4), cysteine-rich (CR) domain, and portion of thecarboxy-terminal (CT) domain from full length human Dp427m dystrophinprotein (SEQ ID NO:25), which are domains minimally required forfunction. The protein sequence of the mini-dystrophin protein isprovided as the amino acid sequence of SEQ ID NO:7, which is encoded bythe human codon-optimized DNA sequence provided as the nucleic acidsequence of SEQ ID NO:1. The vector genome of theAAV9.hCK.Hopti-Dys3978.spA vector is provided as the nucleic acidsequence of SEQ ID NO:18, or its reverse complement when thesingle-stranded genome is packaged in its minus polarity.

The vector genome comprises 5′ and 3′ flanking AAV2 inverted terminalrepeats (ITRs) (having the DNA sequence of SEQ ID NO:14 or SEQ ID NO:15,respectively), a synthetic hybrid enhancer and promoter derived from thecreatine kinase (CK) gene to serve as a muscle specific transcriptionregulatory element (hCK; having the DNA sequence of SEQ ID NO:16), a3978 base pair long human codon-optimized gene encoding the humanmini-dystrophin protein described above (i.e., the Hopti-Dys3978 gene),and a small synthetic transcription termination sequence including apolyadenylation (polyA) signal (spA; having the DNA sequence of SEQ IDNO:17).

Vector was manufactured using the triple transfection technique and aserum free non-adherent cell line derived from HEK 293 cells. Theplasmids used included a helper plasmid to express adenovirus helperproteins required for efficient replication and packaging of the vector,a packaging plasmid expressing the AAV2 rep gene and the AAV9 capsidproteins, and a third plasmid containing the sequence of the expressioncassette described above.

Cells were grown and expanded from a working cell bank sample, and oncesufficient volume and cell density had been reached, the cells weretransfected using a transfection reagent. After incubation to permitvector production from the transfected cells, the cells were lysed torelease vector, the lysate clarified, and vector purified using anuclease treatment step to remove contaminating nucleic acids, followedby iodixanol step gradient centrifugation, anion exchangechromatography, dialysis against the formulation buffer, sterilefiltration, and then storage at 2-8° C.

Example 7 Effects of Single Dose of AAV9.hCK.Hopti-Dys3978.spA in a RatModel of DMD

This example describes testing AAV9.hCK.Hopti-Dys3978.spA in a recentlydeveloped Dmd^(mdx) rat model, which has certain advantages compared tothe classic mdx mouse and GRMD dog models. Larcher, T., et al.,Characterization of dystrophin deficient rats: a new model for Duchennemuscular dystrophy. PLoS One. 2014; 9(10):e110371. In particular, in theDmd^(mdx) rat model, the skeletal and cardiac disease are both presentat an early stage and develop in a sequential manner similar to thedisease progression seen in humans.

In these studies, male Dmd^(mdx) rats 5-6 weeks of age were systemicallyadministered by IV injection into tail veins a single dose (1×10¹⁴vector genomes per kilogram body weight, or vg/kg) of Dys3978 vectorsuspended in PBS. As a control, wild-type (“WT”) rats from the samegenetic background (Sprague Dawley) were also treated in this way. Allprocedures were conducted blinded to the rat genotype or treatmentcohort to avoid bias. Three Dmd^(mdx) rats and 4 WT rats were treatedwith vector, whereas 3 Dmd^(mdx) rats and 2 WT rats were administeredPBS only as a negative control (mock treatment). Three monthspost-injection, animals were euthanized and underwent necropsy fortissue analysis by histology and immunocytochemistry for dystrophinprotein expression.

For histopathological evaluation, tissue samples were fixed in 10%neutral buffered formalin, embedded in paraffin wax, and 5-μm-thicksectioned before staining with hematoxylin eosin saffron (HES). Fordystrophin immunolabelling, additional samples (liver, heart, bicepsfemoris, pectoralis and diaphragm muscles) were frozen and 8-μm-thicksectioned. Mouse monoclonal antibody NCL-DYSB for dystrophin (NovocastraLaboratories, Newcastle on Tyne, UK) was used for both dystrophin andmini-dystrophin protein detection (1:50), since this antibody does notdistinguish between full length wild type dystrophin and the engineeredmini-dystrophin. All necropsies and histological observations wereperformed in blinded fashion.

By histological examination, no lesions were observed in skeletal orcardiac muscle of PBS and vector treated WT rats. In all Dmd^(mdx) rats,skeletal muscle fiber lesions showing individual necrosis, clusters ofregenerative small fibers, scattered giant hyaline fibers, anisocytosis,centronucleation, endomysial fibrosis and sporadic adiposis were presentand characteristic of DMD skeletal muscle. The incidence and intensityof these lesions was globally decreased in Dmd^(mdx) rats treated withvector compared to those treated only PBS. In the heart, lesions ofmultifocal necrosis, mononuclear cell focal infiltration and mild focalextensive fibrosis were present in one of the Dmd^(mdx) rats (rat 49)treated with PBS, which is characteristic of DMID cardiac muscle. In allthe Dmd^(mdx) rats treated with vector, cardiac muscle presentation wassimilar and showed mild mononuclear cell focal infiltration as seen inthe Dmd^(mdx) rat receiving PBS, but in contrast, no fibrotic foci wereobserved in the hearts of the vector treated Dmd^(mdx) rats.

Using immunocytochemistry, WT rats displayed subsarcolemmal dystrophindetected in skeletal, diaphragm and cardiac muscle fibers, andlocalization of dystrophin detected did not differ between rats treatedwith vector compared to only PBS. However, mini-dystrophin detection inthe vector treated WT rats could not be confirmed using this assaybecause the anti-dystrophin antibody used could not distinguish betweenwild type dystrophin and the mini-dystrophin protein. By contrast, oneof the Dmd^(mdx) rats (rat 49) displayed rare skeletal muscle fibers(from about 5% to 10%) with subsarcolemmal dystrophin detectable, whichis in accordance with the previous description of the presence ofscattered revertant fibers in this model with a frequency of about 5%(Larcher et al., PlosOne, 2014). However no dystrophin was detected indiaphragm or cardiac muscle fibers from this rat. In all Dys3978 vectortreated Dmd^(mdx) rats, subsarcolemmal dystrophin was also detected inabout 80% to 95% of skeletal muscle fibers, about 30% to 50% indiaphragm muscle fibers, and about 70% to 80% in heart muscle fibers,although no systematic counting performed. In these rats, very rareskeletal muscle fibers (1 or 2 per muscle section) displayed somecytoplasmic interfibrillar dystrophin. In both vector treated WT andDmd^(mdx) rats, there was no evidence of inflammatory cell infiltratesor increased necrosis that might indicate that a cellular immuneresponse had been stimulated by vector transduction, or production ofthe mini-dystrophin.

In sum, 3 months after systemic administration of 1×10¹⁴ vg/kg ofAAV9.hCK.opti-Dys3978.spA vector, no histological alteration of themuscle tissues was observed in WT rats treated with vector compared toPBS, suggesting that expression of the mini-dystrophin protein was welltolerated in healthy animals. Furthermore, vector treatment of theDmd^(mdx) rats resulted in a significant and generalized detection ofmini-dystrophin in fibers of all muscles studied (biceps femoris,pectoralis, diaphragm and heart) with a pattern of subsarcolemmallocalization similar to that in WT rat muscles. The expression ofmini-dystrophin Dys3978 from the vector was associated with reduction infibrosis and necrosis (FIGS. 36A-36D).

Example 8 Effects of Increasing Doses of AAV9.hCK.Hopti-Dys3978.spA inDmd^(mdx), Rats Determined at 3 Months and 6 Months Post-Injection

This example describes the results of treating Dmd^(mdx) rats, an animalmodel for Duchenne muscular dystrophy, with increasing doses ofAAV9.hCK.Hopti-Dys3978.spA, and measuring the effects at 3 months and 6months after administration.

Rats were dosed at 7-8 weeks of age by IV injection into the dorsalpenile vein, which resulted in systemic administration of the testarticles. Four different vector doses were tested in 10-12 Dmd^(mdx)rats: 1×10¹³ vg/kg (5 rats at the 3 month time point and 6 rats at the 6month time point), 3×10¹³ vg/kg (6 rats at the 3 month time point and 5rats at the 6 month time point), 1×10¹⁴ vg/kg (7 rats at the 3 monthtime point and 6 rats at the 6 month time point), and 3×10¹⁴ vg/kg (5rats at the 3 month time point and 5 rats at the 6 month time point). Inaddition, Dmd^(mdx) rats and WT rats each received vehicle only (1×PBS,215 mM NaCl, 1.25% human serum albumin, 5% (w/v) sorbitol) as a negativecontrol (6 Dmd^(mdx) rats at the 3 month time point, 4 Dmd^(mdx) rats atthe 6 month time point, 5 WT rats at the 3 month time point, and 7 WTrats at the 6 month time point). Five untreated (that is, no vector andno vehicle either) Dmd^(mdx) rats were also included as further negativecontrols. At 3 months and 6 months post-injection, rats from each testarm were euthanized and necropsied to take tissue samples for furtheranalysis. Prior to sacrifice, cardiac function and grip strength testswere carried out in the test animals to assess the effect of vectortreatment on DMD disease progression.

Note that vector doses may be represented in two different numericallyequivalent ways in the text and figures. Thus, “1×10¹³” is equivalent to“1E13,” “3×10¹³” is equivalent to “3E13,” “1×10¹⁴” is equivalent to“1E14,” and “3×10¹⁴” is equivalent to “3E14.”

Body Weight

After treatment and prior to sacrifice, rats in each treatment arm wereweighed daily for the first week, and weekly thereafter until sacrifice.The average weight of all rats in each treatment arm is listed in Table2 (pre-injection until 9 weeks post-injection) and Table 3 (weeks 10-25post-injection) and are graphed against time in FIG. 37 . In the graph,error bars represent the standard error of the mean (SEM), which arealso reported in the table. At all times, the average weight of WT ratsexceeded that of Dmd^(mdx) rats, including those that were treated withvector. Due to age differences and natural variability in body massamong the Dmd^(mdx) rats there was no consistent correlation betweendose and body weight until by 4 weeks post-injection when weights of allvector treated Dmd^(mdx) rats except in the highest dose arm were higherthan untreated Dmd^(mdx) rats, but lower than WT rats. By 12 weekspost-injection, a dose effect in all treatment arms was evident, withbody weight being proportional to vector dose at all doses testedthrough the end of the study.

TABLE 2 MEAN WEIGHTS (g) W-1 D0 D + 1 D + 2 D + 3 D + 4 D + 5 D + 6 D +7 W + 2 W + 3 W + 4 W + 5 W + 6 W + 7 W + 8 W + 9 WT + Buffer 200.0237.9 231.7 237.9 247.1 253.1 262.1 268.7 276.6 325.0 361.8 388.8 410.2428.8 449.6 463.4 482.1 (n = 12 until W + 13, then n = 7) SEM 10.9 10.510.1 10.5 10.1 9.8 10.0 10.4 10.7 10.8 11.7 11.9 13.6 14.8 15.4 15.514.1 DMD + Buffer 180.4 207.4 203.4 208.8 214.5 221.5 229.8 235.4 244.3286.3 318.4 340.5 358.7 374.4 389.1 403.6 418.6 (n = 10 until W + 13,then n = 4) SEM 11.9 12.3 11.9 12.6 12.4 13.0 12.4 13.1 12.6 15.1 17.718.7 19.4 20.3 21.3 21.0 22.3 DMD + 1E13 vg/kg 173.4 206.4 205.8 208.4216.7 223.8 229.8 236.8 243.1 286.3 325.6 346.5 368.6 388.1 403.8 419.5439.1 (n = 11 until W + 13, then n = 6) SEM 10.3 8.3 7.9 7.5 7.9 7.2 7.67.8 8.7 9.5 12.6 13.2 14.2 15.6 16.1 16.7 16.2 DMD + 3E13 vg/kg 180.2210.8 206.3 209.1 217.6 224.9 230.9 237.2 245.2 297.7 329.5 357.3 380.8398.4 416.7 432.9 448.2 (n = 11 until W + 13, then n = 5) SEM 11.9 10.910.3 10.9 11.3 10.5 10.6 10.7 11.0 13.4 13.5 15.3 16.5 17.1 18.0 19.419.3 DMD + 1E14 vg/kg 178.2 209.4 204.3 210.5 213.0 220.1 225.3 232.5244.5 288.2 329.6 356.6 375.9 395.7 413.4 431.5 448.6 (n = 13 until W +13, then n = 6) SEM 9.6 7.9 7.5 7.7 7.5 7.4 7.0 7.5 9.9 9.6 11.7 12.312.2 12.4 12.7 13.8 13.3 DMD + 1E14 230.1 234.9 224.5 233.7 238.7 245.3243.0 252.7 254.9 300.6 331.7 352.7 370.3 382.2 392.3 421.3 428.5 vg/kgw/o HSA (n = 5 until W + 13) SEM 10.2 9.9 9.3 9.3 9.5 9.5 9.3 9.8 10.413.0 15.4 15.8 17.1 18.1 16.3 17.2 22.7 DMD + 3E14 vg/kg 161.8 198.4193.2 197.1 202.3 208.6 212.3 220.6 223.7 272.6 316.2 350.7 373.0 393.3414.9 429.2 445.8 (n = 10 until W + 12, then n = 5) SEM 6.1 7.5 8.8 9.99.3 8.9 8.7 8.7 8.5 8.2 8.7 10.5 11.4 13.9 13.6 14.4 14.7

TABLE 3 MEAN WEIGHTS (g) W + 10 W + 11 W + 12 W + 13 W + 14 W + 15 W +16 W + 17 WT + Buffer 490.7 505.2 509.1 516.8 514.0 527.8 545.0 553.8 (n= 12 until W + 13, then n = 7) SEM 14.0 12.4 13.2 13.3 18.8 19.6 19.218.5 DMD + Buffer 423.0 435.2 428.2 430.3 430.2 440.6 452.7 461.8 (n =10 until W + 13, then n = 4) SEM 22.8 21.8 17.4 21.6 30.5 32.3 37.7 39.4DMD + 1E13 vg/kg 444.8 452.5 453.5 464.5 453.2 457.7 469.9 481.3 (n = 11until W + 13, then n = 6) SEM 16.6 17.3 15.4 17.7 17.4 17.2 18.7 17.6DMD + 3E13 vg/kg 458.2 467.4 469.7 476.1 465.8 478.5 489.1 496.9 (n = 11until W + 13, then n = 5) SEM 20.2 19.4 20.7 23.1 28.9 31.4 34.2 34.2DMD + 1E14 vg/kg 459.3 472.7 478.0 483.2 482.1 492.5 504.5 513.6 (n = 13until W + 13, then n = 6) SEM 12.7 12.3 11.8 12.2 13.7 14.1 14.3 13.0DMD + 1E14 vg/kg w/o HSA 430.2 464.5 469.1 470.3 N/A N/A N/A N/A (n = 5until W + 13) SEM 20.4 18.0 18.7 18.1 N/A N/A N/A N/A DMD + 3E14 vg/kg457.4 475.3 483.9 502.1 515.9 519.1 532.8 547.1 (n = 10 until W + 12,then n = 5) SEM 14.8 15.0 15.6 27.5 26.7 24.9 27.2 26.5 MEAN WEIGHTS (g)W + 18 W + 19 W + 20 W + 21 W + 22 W + 23 W + 24 W + 25 WT + Buffer562.2 572.0 577.2 581.9 586.9 597.3 613.8 596.4 (n = 12 until W + 13,then n = 7) SEM 19.4 18.8 21.6 22.9 24.1 23.1 19.5 26.6 DMD + Buffer464.8 463.5 464.9 463.0 465.8 467.1 467.4 444.8 (n = 10 until W + 13,then n = 4) SEM 39.2 42.7 43.6 46.5 46.6 46.6 46.6 34.9 DMD + 1E13 vg/kg484.8 491.7 490.1 496.3 491.6 513.3 513.3 467.2 (n = 11 until W + 13,then n = 6) SEM 18.3 21.2 23.7 24.2 27.0 35.8 35.8 36.4 DMD + 3E13 vg/kg505.2 510.7 512.6 517.4 519.7 525.7 525.7 507.0 (n = 11 until W + 13,then n = 5) SEM 35.3 39.4 38.9 38.3 37.7 35.6 35.6 30.4 DMD + 1E14 vg/kg525.0 532.7 530.6 541.9 545.9 539.7 539.7 551.3 (n = 13 until W + 13,then n = 6) SEM 14.8 15.4 13.8 14.8 15.4 9.2 9.2 15.1 DMD + 1E14 vg/kgw/o HSA N/A N/A N/A N/A N/A N/A N/A N/A (n = 5 until W + 13) SEM N/A N/AN/A N/A N/A N/A N/A N/A DMD + 3E14 vg/kg 552.6 558.0 558.1 565.9 566.4577.6 577.6 566.9 (n = 10 until W + 12, then n = 5) SEM 27.4 27.2 27.327.3 28.8 29.2 29.2 28.5Quantification of Vector Transduction and RNA and Protein Expression inDmd^(mdx) Rats Treated with AAV9.hCK.Hopti-Dys3978.spA Vector

Materials and Methods

Standard molecular biology techniques were used to quantitate thetransgene copy number by quantitative PCR (qPCR), relative expressionlevels of the mini-dystrophin mRNA transcripts by reverse transcriptaseqPCR (RT-qPCR), and the amount of mini-dystrophin protein expressionqualitatively by Western blot analysis.

For qPCR, genomic DNA (gDNA) was purified from tissues using the GentraPuregene kit from Qiagen. Samples were then analyzed using a StepOnePlus' Real Time PCR System (Applied Biosystems®, Thermo FisherScientific) using 50 ng gDNA in duplicate. All reactions were performedin duplex in a final volume of 20 μL containing template DNA, Premix Extaq (Ozyme), 0.3 μL of ROX reference Dye (Ozyme), 0.2 μmol/L of eachprimer and 0.1 μmol/L of Tagman® probe.

Vector copy numbers were determined using primers and probe designed toamplify a region of the mini-dystrophin transgene:

SEQ ID NO: 19 Forward: 5′-CCAACAAAGTGCCCTACTACATC-3′ SEQ ID NO: 20Reverse: 5′- GGTTGTGCTGGTCCAGGGCGT-3′ SEQ ID NO: 21Probe: 5′-FAM-CCGAGCTGTATCAGAGCCTGGCC-TAMRA-3′

Endogenous gDNA copy numbers were determined using primers and probedesigned to amplify the rat HPRT1 gene:

Forward: SEQ ID NO: 22 5′- GCGAAAGTGGAAAAGCCAAGT -3′ Reverse:SEQ ID NO: 23 5′-GCCACATCAACAGGACTCTTGTAG-3′ Probe: SEQ ID NO: 245′- JOE- CAAAGCCTAAAAGACAGCGGCAAGTTGAAT-TAMRA-3′

For each sample, threshold cycle (Ct) values were compared with thoseobtained with different dilutions of linearized standard plasmids(containing either the mini-dystrophin expression cassette or the ratHPRT1 gene). The absence of qPCR inhibition in the presence of gDNA waschecked by analyzing 50 ng of gDNA extracted from tissues samples from acontrol animal, spiked with different dilutions of standard plasmid.Duplex qPCR (amplification of the 2 sequences in the same reaction) wasused and results were expressed in vector genome per diploid genome(vg/dg). The sensitivity of the test was 0.003 vg/dg.

For RT-qPCR, total RNA was extracted from tissue samples withTRIzol@reagent (Thermo Fisher Scientific), and then treated withRNAse-free DNAse I from the TURBO DNA-free kit (Thermo FischerScientific). Total RNA (500 ng) was reverse transcribed using randomprimers (Thermo Fischer Scientific) and M-MLV reverse transcriptase(Thermo Fischer Scientific) in a final volume of 25 μL. Duplex qPCRanalysis was then performed 1/15-diluted cDNA using the samemini-dystrophin and rat HPRT1 specific primers and probes as for thequantification of transgene copy numbers by qPCR. The absence of qPCRinhibition in the presence of cDNA was checked by analyzing cDNAobtained from tissues samples from a control animal spiked withdifferent dilutions of standard plasmid. For each RNA sample, Ct valueswere compared with those obtained with different dilutions of standardplasmids (containing either the mini-dystrophin expression cassette orthe rat HPRT1 gene). Results were expressed in relative quantities (RQ):

RQ=2^(−ΔCt)=2^(−(Ct target-Ct endogenous control))

For each RNA sample, the absence of DNA contamination was also confirmedby analysis of “cDNA-like samples” obtained without addition of reversetranscriptase in the reaction mix.

For Western blot analysis of expressed protein levels, total proteinswere extracted from tissue samples using RIPA buffer containing aprotease inhibitor cocktail (Sigma-Aldrich). Protein extracts, 50 μg forbiceps femoris, heart and diaphragm, or 100 μg for liver, were loaded ona NuPAGE® Novex 3-8% Tris Acetate gel and analyzed using the NuPAGE®large protein blotting kit (Thermo Fischer Scientific). A finalconcentration of 200 mM DTT was used to reduce proteins before loading.Membranes were then blocked in 5% skim milk, 1% NP40 (Sigma-Aldrich) inTBST (tris-buffered saline, 0.1% Tween 20) and hybridized with ananti-dystrophin antibody specific for exons 10 and 11 of the dystrophinprotein (1:100, MANEX 1011C monoclonal antibody) and with a secondaryanti-mouse IgG HRP-conjugated antibody (1:2000, Dako). For proteinloading control, the same membrane was also hybridized with an anti-ratalpha-tubulin antibody (1:10000, Sigma) and with a secondary anti-mouseIgG HRP-conjugated antibody (1:2000, Dako). Immunoblots were visualizedby ECL Chemiluminescent analysis system (Thermo Fisher Scientific).

Human Mini-Dystrophin Transgene Copy Numbers at 3 and 6 MonthsPost-Injection

Results of testing for transgene copy numbers (as vector genomes perdiploid genome (vg/dg)) in whole blood, spleen, heart, biceps femoris,pectoralis, diaphragm, and liver in Dmd^(mdx) rats treated with vectorand vehicle, and in WT rats administered vehicle only are described inthe tables below. Data at 3 months post-injection is provided in Table4, and at 6 months post-injection is provided in Table 5. Data are themean of results from individual test animals.

TABLE 4 3 Months Post-Injection DMD + DMD + DMD + WT + DMD + 1 × 10¹³ 3× 10¹³ 1 × 10¹⁴ 3 × 10¹⁴ DMD + vehicle vehicle vg/kg vg/kg vg/kg vg/kgWhole blood <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 Spleen <0.002<0.002 <0.002 0.010 0.005 0.013 Heart (basal <0.002 <0.002 0.090 0.2700.670 4.350 part) Biceps <0.002 <0.002 <0.002 0.070 0.260 1.700 femorisPectoralis <0.002 <0.002 0.010 0.030 0.400 0.760 Diaphragm <0.002 <0.0020.003 0.030 2.410 2.810 Liver (central <0.002 <0.002 0.830 5.460 30.780112.880 lobe)

TABLE 5 6 Months Post-Injection DMD + DMD + DMD + WT + DMD + 1 × 10¹³ 3× 10¹³ 1 × 10¹⁴ 3 × 10¹⁴ DMD + vehicle vehicle vg/kg vg/kg vg/kg vg/kgWhole blood <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 Spleen <0.002<0.002 <0.002 <0.002 <0.002 0.010 Heart (basal <0.002 <0.002 0.160 0.1401.460 5.380 part) Biceps <0.002 <0.002 0.009 0.020 0.390 1.400 femorisPectoralis <0.002 <0.002 0.006 0.020 0.530 0.800 Diaphragm <0.002 <0.0020.010 0.010 4.850 1.270 Liver (central <0.002 <0.002 1.080 8.130 30.49082.230 lobe)

No qPCR signal was detected in the Dmd^(mdx) or WT rats injected withvehicle only, confirming that these animals had not received any vector,and no qPCR signal was detected in whole blood at 3 and 6 monthspost-injection.

Mini-dystrophin DNA was detected in Dmd^(mdx) rats that had beeninjected with vector at both 3 and 6 months post-injection. Transgenecopy numbers in the tissues under study followed a pattern of prevalenceof liver >heart >biceps femoris ˜ diaphragm pectoralis >spleen. Of thetissues analyzed, liver was by far the most efficiently transduced, withvector copy numbers reaching up to an average of 80-110 vg/dg in ratsadministered with 3×10¹⁴ vg/kg vector. Vector copy numbers in liver were7-45 fold higher than in heart and 40-300 fold higher than in bicepsfemoris, diaphragm, or pectoralis muscles. In heart, vector copy numbersaveraged about 1.0 vg/dg in rats dosed with 1×10¹⁴ vg/kg vector andabout 5.0 vg/dg in rats dosed with 3×10¹⁴ vg/kg vector. At a dose of1×10¹⁴ vg/kg, transgene copy numbers in biceps femoris and pectoraliswere similar and never exceeded about 0.5 vg/dg. When the vector doseincreased to 3×10¹⁴ vg/kg, the average transgene copy number increasedto about 1.2 vg/dg. The data was particularly variable for diaphragm dueto certain unusually high results among 4 animals that had received thetwo highest dose levels of vector, in which the transgene copy numbersranged from about 9-15 vg/dg. If these outlying data points areexcluded, then the transduction efficiency of diaphragm is relativelylow at both the 3 and 6 month time points, with transgene copy numbersaveraging about 0.2-0.4 vg/dg at the 1×10¹⁴ vg/kg dose and about1.05-1.3 vg/dg at the 3×10¹⁴ vg/kg dose.

Human Mini-Dystrophin mRNA Expression at 3 and 6 Months Post-Injection

Two to four animals per treatment arm were randomly chosen for analysisby RT-qPCR to quantify levels of human mini-dystrophin mRNA transcriptsin samples of biceps femoris, diaphragm, heart, spleen, and liverobtained at sacrifice. The results obtained from test animals sacrificedat 3 months and 6 months post-injection are provided in Table 6 andTable 7, respectively. Data is expressed in relative quantities (RQ) ofmini-dystrophin mRNA relative to mRNA from the rat HPRT1 gene.

No transcripts were detected in any tissue from animals in the negativecontrol arms (WT rats and Dmd^(mdx) rats treated with vehicle), or inspleen of animals treated with vector, regardless of dose. In all othertissues examined, vector-derived transcripts were detected, the levelsof which tended to increase in a dose-responsive manner, although withsome variability in the data. Transcript levels in the tissues followedthe pattern biceps femoris >heart ˜ diaphragm >liver. As discussedabove, liver was the most transduced tissue among those sampled, withvector copy numbers varying about 60-130 fold higher than in bicepsfemoris muscle. Despite this, the level of mini-dystrophin mRNA in liverwas about 5-15 fold lower than in biceps femoris, evidence of the highlymuscle-specific activity of the promoter used in the vectors.

TABLE 6 3 Months Post-Injection Rat 5 Rat 6 Rat 7 Rat 8 Rat 9 Rat 10 Rat11 Rat 12 Rat 13 Rat 14 Rat 1 Rat 2 Rat 3 Rat 4 RQ RQ RQ RQ RQ RQ RQ RQRQ RQ RQ RQ RQ RQ DMD + DMD + DMD + DMD + DMD + DMD + DMD + DMD + DMD +DMD + DMD + DMD + WT + WT + 1 × 10¹³ 1 × 10¹³ 3 × 10¹³ 3 × 10¹³ 1 × 10¹⁴1 × 10¹⁴ 1 × 10¹⁴ 1 × 10¹⁴ 3 × 10¹⁴ 3 × 10¹⁴ vehicle vehicle vehiclevehicle vg/kg vg/kg vg/kg vg/kg vg/kg vg/kg vg/kg vg/kg vg/kg vg/kgSpleen <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03<0.03 <0.03 <0.03 Biceps <0.03 <0.03 <0.03 <0.03 2.6 0.9 7.8 7.2 23.818.4 40.9 79.6 33.1 33.3 femoris Heart <0.03 <0.03 <0.03 <0.03 1.7 1.83.3 1.4 4.5 4.5 3.2 6.5 9.6 12.4 (basal part) Diaphragm <0.03 <0.03<0.03 <0.03 0.2 0.3 1.6 2.7 13.6 5.1 4.2 23.2 9.7 18.8 Liver <0.03 <0.03<0.03 <0.03 0.1 0.2 0.7 0.8 2.2 4.7 3.8 0.8 7.4 3.3 (central lobe)

TABLE 7 6 Months Post-Injection Rat 19 Rat 20 Rat 21 Rat 22 Rat 23 Rat24 Rat 25 Rat 26 Rat 15 Rat 16 Rat 17 Rat 18 RQ RQ RQ RQ RQ RQ RQ RQ RQRQ RQ RQ DMD + DMD + DMD + DMD + DMD + DMD + DMD + DMD + DMD + DMD +WT + WT + 1 × 10¹³ 1 × 10¹³ 3 × 10¹³ 3 × 10¹³ 1 × 10¹⁴ 1 × 10¹⁴ 3 × 10¹⁴3 × 10¹⁴ vehicle vehicle vehicle vehicle vg/kg vg/kg vg/kg vg/kg vg/kgvg/kg vg/kg vg/kg Spleen <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03<0.03 <0.03 <0.03 <0.03 Biceps <0.03 <0.03 <0.03 <0.03 0.6 0.3 3.0 8.915.8 24.2 64.0 19.7 femoris Heart (basal <0.03 <0.03 <0.03 <0.03 1.2 1.61.3 1.4 3.7 4.3 9.2 6.1 part) Diaphragm <0.03 <0.03 <0.03 <0.03 0.5 0.11.4 1.1 4.5 8.0 19.7 17.1 Liver (central <0.03 <0.03 <0.03 <0.03 0.1 0.10.2 0.5 0.7 0.6 4.6 2.1 lobe)

Human Mini-Dystrophin Protein Expression at 3 and 6 MonthsPost-Injection

The same animals randomly selected for analysis to determine humanmini-dystrophin mRNA levels were also analyzed to determinemini-dystrophin protein levels using Western blot. No mini-dystrophinprotein was detected in any tissue from animals in the negative controlarms (WT rats and Dmd^(mdx) rats treated with vehicle). At both the 3and 6 month time points, mini-dystrophin protein was detected in bicepsfemoris, heart and diaphragm of Dmd^(mdx) rats dosed with vector. At thelowest dose tested (1×10¹³ vg/kg), mini-dystrophin protein was detectedless frequently in the tissue samples compared to rats dosed with vectorat higher levels. These results are summarized qualitatively in Table 8.

TABLE 8 Heart Biceps (basal Rat Time Dose femoris part) Diaphragm  5 3mo 1 × 10¹³ vg/kg + + +  6 3 mo 1 × 10¹³ vg/kg − + −  7 3 mo 3 × 10¹³vg/kg + + +  8 3 mo 3 × 10¹³ vg/kg + + +  9 3 mo 1 × 10¹⁴ vg/kg + + + 103 mo 1 × 10¹⁴ vg/kg + + + 11 3 mo 1 × 10¹⁴ vg/kg + + + 12 3 mo 1 × 10¹⁴vg/kg + − + 13 3 mo 3 × 10¹⁴ vg/kg + + + 14 3 mo 3 × 10¹⁴ vg/kg + + + 196 mo 1 × 10¹³ vg/kg + + − 20 6 mo 1 × 10¹³ vg/kg − + − 21 6 mo 3 × 10¹³vg/kg + + + 22 6 mo 3 × 10¹³ vg/kg + + − 23 6 mo 1 × 10¹⁴ vg/kg + + + 246 mo 1 × 10¹⁴ vg/kg + + + 25 6 mo 3 × 10¹⁴ vg/kg + + + 26 6 mo 3 × 10¹⁴vg/kg + + +

There was a positive correlation between the amount of protein detectedby Western blot and the vector dose, as well as the amount ofmini-dystrophin mRNA in the same tissue samples. A mini-dystrophin mRNARQ of approximately 1.5 was required to permit detection of the protein.Consistent with the low levels of mini-dystrophin transcript measured inliver, no mini-dystrophin protein was detected in this tissue, even atthe highest vector dose used.

Histopathological Assessment

Immediately after sacrifice of WT and Dmd^(mdx) rats, tissue sampleswere obtained for histopathological and immunocytochemical analysis.

Materials and Methods

Tissue samples vehicle treated WT rats, vehicle and vector treatedDmd^(mdx) rats were obtained during whole necropsy evaluation at 3 and 6months post-injection. Samples were also obtained from untreatedDmd^(mdx) rats sacrificed at 7-9 weeks of age to serve as a baselinecomparison. Tissues were immediately fixed in formalin forhistopathology or snap frozen for immunohistochemistry (immunolabeling)and stored until processing. For histopathology, tissue samples werefixed in 10% neutral buffered formalin, embedded in paraffin wax, andsectioned (5 m) before staining with hematoxylin eosin saffron (HES)stain. An additional section of paraffin embedded heart tissue wasstained to visualize collagen with picrosirius red F3B (Sigma-AldrichChimie SARL, Lyon, FR). To identify dystrophin and connective tissue byimmunolabeling, samples were frozen and sectioned (8 m). A mousemonoclonal antibody, NCL-DYSB (1:50, Novocastra Laboratories, Newcastleon Tyne, UK), that specifically binds to rat dystrophin as well as humanmini-dystrophin opti-Dys3978 was used in immunolabeling studies tovisualize dystrophin protein. Alexa Fluor 555 wheat germ agglutinin(WGA) conjugate (1:500, Molecular Probes, Eugene, OR) was used tovisualize connective tissue. Nuclei were stained with DRAQ5 (1:1000,BioStatus Ltd, Shepshed, UK). Necropsies and histological examinationwere performed blinded.

Quantification of the picrosirius positive areas in heart sections wasperformed using Nikon Imaging Software (Nikon, Champigny sur Marne,France). Quantification of DYSB positive fibers and WGA positive areaswas performed using ImageJ open source image processing software (v2.0.0-rc-49/1.51a).

Results

Histopathological Analysis of DMD Lesions in Muscle at 3 and 6 MonthsPost-Injection

Tissue samples stained for histology were examined microscopically andlesions related to the DMD phenotype systematically recorded. Lesions inskeletal and cardiac muscle were scored semi-quantitatively asillustrated in FIG. 38A. In skeletal muscle (biceps femoris, pectoralisand diaphragm), a score of 0 corresponded to absence of significantlesion; a score of 1 corresponded to the presence of some regenerationactivity as evidenced by centro-nucleated fibers and regeneration foci;a score of 2 corresponded to degenerative fibers, isolated or in smallclusters; and a score of 3 corresponded to tissue remodeling and fiberreplacement by fibrotic or adipose tissue. In the heart, scoring wasbased on the intensity of fibrosis (score of 1 for lower, and score of 2for higher) and the presence of degenerative fibers (score of 3). Atotal lesion score for each rat was calculated as the mean of theanimal's scores for biceps femoris, pectoralis, diaphragm and cardiacmuscles. Lesion scores for individual rats within each treatment armwere also averaged.

Total lesion scores of individual rats and averages grouped by treatmentarm at 3 months post-injection are shown in FIG. 38B, in which WT mockrefers to WT rats treated with vehicle, for which lesion scores were 0.KO mock refers to Dmd^(mdx) rats treated with vehicle, whereas KO 1E13,3E13, and 1E14, refer to Dmd^(mdx) rats treated with the indicated doses(i.e., 1×10¹³, 3×10¹³, and 1×10¹⁴, respectively) of vector in vg/kg. Ascan be seen, the prevalence of muscular lesions associated with thedystrophic phenotype in Dmd^(mdx) rats was reduced by vector treatmentin a dose-responsive manner.

Statistical analysis of lesion scores (by multiple paired comparisonsusing Dunn's test) revealed the following differences among treatmentarms. In samples of biceps femoris muscles at 3 months post-injection,there were no significant differences in lesion scores between WT ratstreated with vehicle and Dmd^(mdx) rats treated with vector at the twohighest doses (1×10¹⁴ and 3×10¹⁴ vg/kg) and at 6 months post-injection,there were no significant differences between WT treated with vehicleand Dmd^(mdx) rats treated with vector at any of the four doses tested.In samples of pectoralis muscle and diaphragm at 3 months post-injectionthere were no significant differences in lesion scores between vehicletreated WT rats and Dmd^(mdx) rats treated with the three highest vectordoses tested (3×10¹³, 1×10¹⁴ and 3×10¹⁴ vg/kg) and at 6 monthspost-injection, there were no significant differences in scores betweenWT rats treated with vehicle and Dmd^(mdx) rats treated with all fourvector doses. Finally, in heart muscle, at both time points, there wereno significant differences in lesion scores between vehicle treated WTrats and Dmd^(mdx) rats treated with all four doses of vector.

Histomorphometry at 3 and 6 Months Post-Injection

After labeling tissue samples with the DYSB antibody, which specificallybinds to both rat dystrophin and the human mini-dystrophin expressedfrom the vector, the percentage of positively stained muscle fibers inthree randomly selected microscopic fields from each rat was calculatedfor biceps femoris, diaphragm, and cardiac muscles. In addition, thearea in three randomly selected microscopic fields staining positivelywith WGA conjugate was calculated to determine the extent of connectivetissue fibrosis in frozen tissue samples from biceps femoris anddiaphragm. In a related analysis, the amount of connective tissue(collagen) in transverse sections of heart was determined by quantifyingthe area staining positive with picrosirius red in histologicalpreparations. Results from these studies are provided in FIGS. 39A-39C,FIGS. 40A-40C, and FIGS. 41A-41C.

FIG. 39A shows representative photomicrographs of stained tissuesections from biceps femoris muscle samples from WT rats treated withvehicle (WT+buffer), Dmd^(mdx) rats treated with vehicle (DMD+buffer),and Dmd^(mdx) rats treated with vector at increasing doses of 1×10¹³,3×10¹³, 1×10¹⁴ and 3×10¹⁴ vg/kg (DMD+1E13, 3E13, 1E14, and 3E14,respectively). The top panel of photos are from samples taken at 3months post-injection and the bottom panel are from samples taken at 6months post-injection. FIG. 39B is a graph showing the percentage ofdystrophin positive fibers in biceps femoris muscle samples from WT ratsand Dmd^(mdx) rats, each treated with vehicle, and Dmd^(mdx) ratstreated with increasing doses of vector, at 3 and 6 month time points.Also included are results from untreated Dmd^(mdx) rats 7-9 weeks of age(“DMD pathol status”). FIG. 39C is a graph showing the percentage areaoccupied by connective tissue (as a measure of fibrosis) in bicepsfemoris muscle samples from similarly treated WT and Dmd^(mdx) rats at 3and 6 month time points, and untreated Dmd^(mdx) rats 7-9 weeks of age.In the graphs, the same letter over error bars indicates nostatistically significant difference between the data, whereas no commonletter indicates there is a significant difference (for example, twobars both having an “a” above them would not be significantly differentfrom each other).

FIG. 40A shows representative photomicrographs of stained tissuesections from diaphragm samples from WT rats treated with vehicle(WT+buffer), Dmd^(mdx) rats treated with vehicle (DMD+buffer), andDmd^(mdx) rats treated with vector at increasing doses of 1×10¹³,3×10¹³, 1×10¹⁴ and 3×10¹⁴ vg/kg (DMD+1E13, 3E13, 1E14, and 3E14,respectively), all taken at 3 months post-injection. FIG. 40B is a graphshowing the percentage of dystrophin positive fibers in diaphragmsamples from WT rats and Dmd^(mdx) rats, each treated with vehicle, andDmd^(mdx) rats treated with increasing doses of vector, at 3 and 6 monthtime points. Also included are results from untreated Dmd^(mdx) rats 7-9weeks of age (“DMD pathol status”). FIG. 40C is a graph showing thepercentage area occupied by connective tissue (as a measure of fibrosis)in diaphragm samples from similarly treated WT and Dmd^(mdx) rats at 3and 6 month time points, and untreated Dmd^(mdx) rats 7-9 weeks of age.In the graphs, the same letter over error bars indicates nostatistically significant difference between the data, whereas no commonletter indicates there is a significant difference (for example, twobars both having an “a” above them would not be significantly differentfrom each other).

FIG. 41A shows representative photomicrographs of stained tissuesections from heart muscle samples from WT rats treated with vehicle(WT+buffer), Dmd^(mdx) rats treated with vehicle (DMD+buffer), andDmd^(mdx) rats treated with vector at increasing doses of 1×10¹³,3×10¹³, 1×10¹⁴ and 3×10¹⁴ vg/kg (DMD+1E13, 3E13, 1E14, and 3E14,respectively). The top and bottom panels show transverse sections ofhearts from the third of the apex prepared histologically and stainedwith picrosirius red taken from test animals sacrificed at 3 and 6months post-injection, respectively. The black bars indicate length of 2mm. The middle panel shows immunolabeling with anti-dystrophin antibodyand WGA conjugate in heart muscle samples taken at the 3 month timepoint. FIG. 41B is a graph showing the percentage of dystrophin positivefibers in heart muscle samples from WT rats and Dmd^(mdx) rats, eachtreated with vehicle, and Dmd^(mdx) rats treated with increasing dosesof vector, at 3 and 6 month time points. Also included are results fromuntreated Dmd^(mdx) rats 7-9 weeks of age (“DMD pathol status”). FIG.41C is a graph showing the percentage area occupied by connective tissue(as a measure of fibrosis) in heart muscle samples from similarlytreated WT and Dmd^(mdx) rats at 3 and 6 month time points, anduntreated Dmd^(mdx) rats 7-9 weeks of age. In the graphs, the sameletter over error bars indicates no statistically significant differencebetween the data, whereas no common letter indicates there is asignificant difference (for example, two bars both having an “a” abovethem would not be significantly different from each other).

Statistical analysis (ANOVA analysis and Fisher's post-hoc bilateraltest) of the data demonstrated that at both 3 and 6 monthspost-injection, there was a significant difference in dystrophinlabeling in biceps femoris and heart between Dmd^(mdx) rats treated withvehicle and Dmd^(mdx) rats treated at all vector doses. In diaphragm,differences at 3 months post-injection were significant at the twohighest doses tested, whereas at 6 months post-injection, thedifferences were significant at the three highest doses tested.Comparison between WT rats treated with vehicle and Dmd^(mdx) ratstreated with 3×10¹⁴ vg/kg revealed no significant difference in bicepsfemoris muscle at 3 months post-injection or in cardiac muscle at 6months post-injection.

In muscles from WT rats treated with vehicle, all muscle fibersdisplayed intense homogeneous subsarcolemmal labeling with the DYSBantibody. In muscles from Dmd^(mdx) rats treated with vehicle, a smallpercentage of scattered revertant fibers displayed similar labeling (at3 and 6 months post-injection, respectively: biceps femoris, 3.7±2.4%and 7.3±2.3%; diaphragm, 0.7±1.5% and 5.8±1.3%; cardiac muscle, 0.0±0.0%and 0.1±0.1%). In Dmd^(mdx) rats administered vector, the percentage offibers staining positive for dystrophin was increased in all observedmuscles with fibers displaying weak to intense subsarcolemmal labeling.Labeling of two thirds of the fiber was required to be consideredpositive. At both 3 and 6 month time points, the percentage ofdystrophin-positive fibers was similar between biceps femoris andcardiac muscle, which was higher than in diaphragm. In Dmd^(mdx) ratstreated with vector, the number and size of the fibrotic foci measuredby the area occupied by connective tissue was reduced in skeletalmuscle, and the intensity of fibrosis decreased in heart muscle.

In untreated Dmd^(mdx) rats sacrificed at 7-9 weeks of age, no fibrosiswas evident in biceps femoris or heart muscle, but there was alreadysignificant connective tissue expansion in diaphragm. Compared to WTrats, vehicle treated Dmd^(mdx) rats displayed focal or generalizedthickening of the endomysial and perimysial space in skeletal muscle,which is indicative of fibrosis. In the heart, these rats displayedscattered and extensive fibrotic foci in ventricular and septalsubepicardial regions. In severe cases, transmural fibrosis was observedthat altered the shape of the heart. Compared with Dmd^(mdx) ratstreated with vehicle, there was a significant reduction in the numberand size of fibrotic foci at 3 months post-injection in the bicepsfemoris of Dmd^(mdx) rats treated with 3×10¹³ vg/kg vector and higherdoses, and at 6 months post-injection in the diaphragm of Dmd^(mdx) ratstreated with 3×10¹⁴ vg/kg vector. In heart, significant differences infibrosis were found between Dmd^(mdx) rats treated with vehicle andDmd^(mdx) rats treated at all vector doses at both time points. At 3months post-injection, no significant difference in fibrosis wasobserved between WT rats treated with vehicle and Dmd^(mdx) rats treatedwith vector at a dose of 3×10¹³ vg/kg and higher. The amount of fibrosisobserved and vector dose were negatively correlated (p=0.019 for bicepsfemoris; p=0.004 for diaphragm; and p=0.003 for cardiac muscle, all bylinear regression).

In Dmd^(mdx) rats treatment with vector induced mini-dystrophinexpression in all muscles analyzed (biceps femoris, diaphragm, andheart), and the percentage of fibers expressing mini-dystrophin waspositively correlated with vector dose (p<0.001 by linear regression).The number of mini-dystrophin-positive fibers in vector treatedDmd^(mdx) rats was higher in biceps femoris and heart than in diaphragm,suggesting some heterogeneity in biodistribution or expression efficacy.Mini-dystrophin expression was similar in terms of its subsarcolemmallocalization, regardless dose, and no abnormal localization was detectedeven at the highest dose analyzed, 3×10¹⁴ vg/kg. In some fibers,discontinuous dystrophin staining was detected along the sarcolemma,although the frequency of this observation decreased with increasingvector dose.

Comparison of the number of mini-dystrophin positive muscle fibersbetween 3 and 6 months post-injection revealed no significantdifferences among treatment arms for biceps femoris. In diaphragm, therewas a significant increase between 3 and 6 months post-injection at the1×10¹⁴ vg/kg dose, whereas in heart muscle, there was a significantincrease between the two time points at the doses 1×10¹³, 3×10¹³, and1×10¹⁴ vg/kg.

The incidence and degree of certain classic DMD related muscle lesionsvaried among the treatment groups. For example, there were fewernecrotic or degenerative fibers vector treated Dmd^(mdx) rats comparedto those that received only vehicle, and newly regenerated fibers wereobserved in all Dmd^(mdx) rats, but their number tended to decrease asvector dose was increased.

Grip Force and Muscle Fatigue Measurements

Forelimb grip force of Dmd^(mdx) rats injected with vehicle orincreasing doses of vector were tested 3 and 6 months post-injection. WTrats injected with vehicle were included as negative controls. Rats wereinjected when they were 7-9 weeks old so that grip force testing wasconducted when they were about 4.5 and 7.5 months old. Maximum gripforce and grip force after repeated trials as an indication of fatiguewere both measured.

Materials and Methods

A grip meter (Bio-GT3, BIOSEB, France) attached to a force transducerwas used to measure the peak force generated when rats were placed withtheir forepaws on the T-bar and gently pulled backward until theyreleased their grip. Five tests were performed in sequence with a shortlatency (20-40 seconds) between each test, and the reduction in strengthbetween the first and the last determination taken as an index offatigue. Results are expressed in grams (g) and are normalized to thebody weight (g/g BW). Grip test measurements were performed by anexperimenter blind to genotype and treatment arm. Data are presented asthe mean±SEM, and evaluated statistically using the non-parametricKruskal-Wallis test to analyze differences between groups. Wheresignificant overall effects were detected, differences between groupswere assessed using Dunn's post-hoc test. Evolution of grip force wasanalyzed using the Friedman test, followed by Dunn's post-hoc test. Alldata analyses were performed using GraphPad Prism 5 (GraphPad SoftwareInc., La Jolla, CA). In figures, significant differences at confidencelevels of 95%, 99%, and 99.9% are represented by one, two and threesymbols, respectively.

Results

Results of grip force tests for rats sacrificed at 3 monthspost-injection are provided in Table 9 and Table 10. As shown in Table9, vehicle treated Dmd^(mdx) rats exhibited a reduction in absolute gripstrength (i.e., not corrected for body mass differences) compared tovehicle treated WT rats (decrease of 24±2%). By contrast, Dmd^(mdx) ratsthat were treated with vector exhibited a dose-dependent increase inabsolute grip strength compared to vehicle treated Dmd^(mdx) ratcontrols. At the two lowest doses, 1×10¹³ and 3×10¹³ vg/kg, grip forceincreased by 13±7% and 24±8%, respectively, but did not reachstatistical significance, while at the two highest doses, 1×10¹⁴ and3×10¹⁴ vg/kg, grip force increased by 40±9% and 55±6%, respectively,which did reach statistical significance (p<0.01 and p<0.001,respectively). Also as shown in Table 9, when forelimb grip force wascorrected for differences in body mass, there was no statisticallysignificant difference between grip force of WT and Dmd^(mdx) rats whenboth were treated with vehicle. However, there was a dose responsiveincrease in relative grip force of vector treated Dmd^(mdx) ratscompared with Dmd^(mdx) rats treated with vehicle, which reachedstatistical significance at the two highest doses tested, 1×10¹⁴ and3×10¹⁴ vg/kg (27±8% increase, p<0.05, and 39±6% increase, p<0.001,respectively).

Forelimb grip force was also measured during five closely spacedrepeated trials to determine the extent to which vector treatment mightaffect the muscle fatigue known to occur in the Dmd^(mdx) rat model. Asshown in FIG. 42A, vehicle treated Dmd^(mdx) rats exhibited a markeddecrease of forelimb strength between the first and fifth trials(reduction of 63±5%), whereas WT rats treated with vehicle were just asstrong after the fifth trial as after the first, an effect seen beforein this model (Larcher, et al., 2014).

In contrast, a dose-dependent improvement was observed in vector treatedDmd^(mdx) rats compared to similar rats treated only with vehicle. Asindicated in Table 10, at the two lowest doses tested (1×10¹³ and 3×10¹³vg/kg) there was delay before a decrease in grip strength manifested,suggesting a reduction in fatigue, at least early in the trials.However, at the lower doses, by the fifth trial, there was still not astatistically significant difference between grip strength of the vectortreated Dmd^(mdx) rats and Dmd^(mdx) rats treated only with vehicle.Nevertheless, a strong trend toward waning reduction in grip strengthwas apparent even at these lower doses. At the two highest doses, 1×10¹⁴and 3×10¹⁴ vg/kg, the Dmd^(mdx) rats showed no statistically significantdifference in the extent of fatigue compared to WT rats treated withvehicle. In other words, after five trials, these vector treatedDmd^(mdx) rats were indistinguishable from wild type. In fact, in alltrials, the mean grip force of Dmd^(mdx) rats treated with the highestvector dose was higher than that of WT controls, although the differencewas not statistically significant.

Results of grip force tests for rats sacrificed at 6 monthspost-injection are provided in Table 11 and Table 12. As shown in Table11, vehicle treated Dmd^(mdx) rats exhibited a reduction in gripstrength (i.e., not corrected for body mass differences) compared tovehicle treated WT rats (decrease of 38±3% in absolute grip force). Thisdifference was statistically significant when measured in absoluteterms, but not when measured in relative terms. By contrast, Dmd^(mdx)rats that were treated with vector exhibited a dose-dependent increasein absolute grip strength compared to vehicle treated Dmd^(mdx) ratcontrols. At the two lowest doses, 1×10¹³ and 3×10¹³ vg/kg, grip forceincreased by 20±5% and 21±6%, respectively, but did not reachstatistical significance, while at the two highest doses, 1×10¹⁴ and3×10¹⁴ vg/kg, grip force increased by 39±9% and 41±5%, respectively,which did reach statistical significance (p<0.05 and p<0.01,respectively).

Similar to the Dmd^(mdx) rats sacrificed 3 months after injection,vehicle treated Dmd^(mdx) rats sacrificed at 6 months post-injectionalso exhibited a substantial decrease of forelimb strength between thefirst and fifth trials (reduction of 57±3%) (FIG. 42B), although thisdifference was not statistically significant compared to the slightreduction in grip force over five trials seen with WT rats treated withvehicle, most likely due to the small sample sizes involved in thesestudies.

In contrast, a dose-dependent improvement was observed in vector treatedDmd^(mdx) rats compared to similar rats treated only with vehicle. Asindicated in Table 12, while the two lowest doses (1×10¹³ and 3×10¹³vg/kg) did not significantly impact the decline in grip strength overmultiple trials, at the two highest doses (1×10¹⁴ and 3×10¹⁴ vg/kg), theDmd^(mdx) rats showed no statistically significant difference in theextent of fatigue compared to WT rats treated with vehicle. Further, atthe highest dose, the grip force of vector treated Dmd^(mdx) rats wasstatistically significantly higher than Dmd^(mdx) rats treated withvehicle at every trial. In other words, after five trials, these vectortreated Dmd^(mdx) rats were indistinguishable from wild type. In fact,in all trials, the mean grip force of Dmd^(mdx) rats treated with thehighest vector dose was higher than that of WT controls, although thedifference was not statistically significant.

Based on these studies, it is evident that at both 3 and 6 monthspost-injection, a vector dose of 1×10¹⁴ vg/kg was sufficient to reversethe reduction in grip force exhibited by Dmd^(mdx) rats and the musclefatigue caused by multiple closely spaced grip force tests. Furthermore,a vector dose of 3×10¹⁴ vg/kg actually improved grip force and fatigueresistance in the Dmd^(mdx) rats to a level that exceeded WT rats of thesame genetic background.

TABLE 9 Grip Force at 4.5 Months of Age (3 Months Post-Injection)Genotype WT DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx) Treatment— — AAV9- AAV9- AAV9- AAV9- optidys3978 optidys3978 optidys3978optidys3978 Dose (vg/kg) — — 1E+13 3E+13 1E+14 3E+14 Body  510.0 ± 12.2 438.1 ± 22.0*  462.7 ± 16.2  469.1 ± 21.0  477.2 ± 11.3  482.9 ± 15.8weight (g) Maximum forelimb grip force g 1743.1 ± 77.2 1318.8 ± 41.8*1493.6 ± 87.3 1640.1 ± 102.7 1848.5 ± 124.8^(□□) 2044.2 ± 83.1^(□□) g/gBW   3.43 ± 0.15   3.06 ± 0.14   3.25 ± 0.19   3.50 ± 0.19   3.87 ±0.24^(□)   4.24 ± 0.13*^(□) n 12 10 11 11 11 10 Animal body weight (g);maximum absolute forelimb grip force (g); and relative forelimb gripforce (g/g of body weight) Values are mean ± SEM n: number of animalstested *p < 0.05 vs WT ¤: p < 0.05, ¤¤: p < 0.01 vs Dmd^(mdx) treatedwith vehicle

TABLE 10 Grip Force Fatigue at 4.5 Months of Age (3 MonthsPost-Injection) Genotype WT DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx)DMD^(mdx) Treatment — — AAV9- AAV9- AAV9- AAV9- optidys3978 optidys3978optidys3978 optidys3978 Dose (vg/kg) — — 1E+13 3E+13 1E+14 3E+14Relative forelimb grip force g/g BW Trial 1 2.98 ± 0.23   2.97 ± 0.14  3.14 ± 0.16   3.16 ± 0.18  3.35 ± 0.31  3.65 ± 0.13^(□) Trial 2 2.92 ±0.21   2.44 ± 0.31^(§)  2.868 ± 0.23    2.9 ± 0.25  3.35 ± 0.29  3.70 ±0.13^(□□) Trial 3 2.89 ± 0.20   1.79 ± 0.26^(§§§)   2.52 ± 0.31^(§)  3.02 ± 0.28^(□)  3.07 ± 0.28^(□)  3.66 ± 0.26^(□□□) Trial 4 3.09 ±0.16   1.45 ± 0.24**^(§§§)   1.81 ± 0.27*^(§§§)   2.32 ± 0.23^(§§§) 3.14 ± 0.31^(□□)  3.84 ± 0.24^(□□□) Trial 5 3.08 ± 0.20   1.10 ±0.17***^(§§§)   1.66 ± 0.18**^(§§§)   2.12 ± 0.25^(§§§)  2.82 ±0.26^(□□□)  3.59 ± 0.22^(□□□) Total decrease 6.13 ± 5.21 −63.53 ±5.49*** −46.65 ± 5.88*** −33.23 ± 7.04* −7.35 ± 11.83^(□□) −1.70 ±4.95^(□□□) Trial 5 vs Trial 1 (% Trial 1) n 11 10 11 11 11 10 Relativeforelimb grip force (g/g of body weight) and decrease in grip forcebetween 1st and 5th trials expressed as percent decrease from 1st trialValues are mean ± SEM n: number of animals tested *p < 0.05, **p < 0.01,***p < 0.001 vs WT ¤: p < 0.05, ¤¤: p < 0.01, ¤¤¤: p < 0.001 vsDmd^(mdx) treated with vehicle ^(§)p < 0.05, ^(§§§)p < 0.001 vs 1sttrial

TABLE 11 Grip Force at 7.5 Months of Age (6 Months Post-Injection)Genotype WT DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx) Treatment— — AAV9- AAV9- AAV9- AAV9- optidys3978 optidys3978 optidys3978optidys3978 Dose (vg/kg) — — 1^(E)+13 3^(E)+13 1^(E)+14 3^(E)+14 Bodyweight (g) 601. ± 24.3  464.7 ± 48.8* 502.6 ± 29.1 527.6 ± 38.0  556.1 ±14.4    577.6 ± 29.2  Maximum forelimb grip force g 2142.7 ± 98.0  1324.0 ± 73.6* 1760.0 ± 150.7 1825.4 ± 72.8    2223.8 ± 122.9^(□□)2350.0 ± 134.1^(□) g/g BW 3.59 ± 0.21  2.90 ± 0.17  3.48 ± 0.16 3.50 ±0.16 4.02 ± 0.26^(□)   4.07 ± 0.15^(□□) n 7 4 6 6 6 5 Animal body weight(g); maximum absolute forelimb grip force (g); and relative forelimbgrip force (g/g of body weight) Values are mean ± SEM n: number ofanimals tested *p < 0.05 vs WT ¤¤: p < 0.01; ¤¤¤: p < 0.001 vs Dmd^(mdx)treated with vehicle

TABLE 12 Grip Force Fatigue at 7.5 Months of Age (6 MonthsPost-Injection) Genotype WT DMD^(mdx) DMD^(mdx) DMD^(mdx) DMD^(mdx)DMD^(mdx) Treatment — — AAV9- AAV9- AAV9- AAV9- optidys3978 optidys3978optidys3978 optidys3978 Dose (vg/kg) — — 1E+13 3E+13 1E+14 3E+14Relative forelimb grip force g/g BW Trial 1   3.48 ± 0.23   2.86 ± 0.18  3.27 ± 0.23   3.44 ± 0.20  3.60 ± 0.32  4.00 ± 0.14^(□□) Trial 2  3.22 ± 0.30   2.58 ± 0.27   3.07 ± 0.20   3.21 ± 0.12  3.31 ± 0.34 3.78 ± 0.12^(□) Trial 3   3.45 ± 0.19   2.07 ± 0.26*^(§§)   2.46 ±0.32^(§)   2.40 ± 0.35^(§§)  3.72 ± 0.33^(□)  3.66 ± 0.14^(□) Trial 4  3.01 ± 0.16   1.52 ± 0.17*^(§§§)   2.19 ± 0.27^(§§)   1.83 ±0.14^(§§§)  3.00 ± 0.38  3.76 ± 0.20^(□□□) Trial 5   3.01 ± 0.16   1.24± 0.14*^(§§§)   1.87 ± 0.33§^(§§§)   1.63 ± 0.16^(§§§)  2.91 ± 0.51^(□) 3.54 ± 0.21^(§□□) Total decrease trial 5 −12.27 ± 5.56 −56.61 ± 3.52 −39.55 ± 13.94 −50.81 ± 8.11   −19.48 ± 11.88 −11.11 ± 5.69 vs Trial 1(% Trial 1) n 7 4 6 5 6 5 Relative forelimb grip force (g/g of bodyweight) and decrease in grip force between 1st and 5th trials expressedas percent decrease from 1st trial Values are mean ± SEM n: number ofanimals tested *p < 0.05 vs WT ¤: p < 0.05, ¤¤: p < 0.01, ¤¤¤: p < 0.001vs Dmd^(mdx) treated with vehicle ^(§)p < 0.05, ^(§§)p < 0.01, ^(§§§)p <0.001 vs 1st trial

Cardiac Function

Cardiac function of Dmd^(mdx) rats and WT controls were tested 3 and 6months post-injection (about 5 and 8 months of age, respectively) todetermine if vector treatment could improve the structural or functionaleffects on heart of the muscular dystrophy disease process in the ratDMD model. Using two-dimensional echocardiography, free wall diastolicthickness, LV end-diastolic diameter, LV ejection fraction, and E/Aratio were measured 3 and 6 months post-injection.

Materials and Methods

Echocardiographic measurements were conducted by an experimenter blindas to genotype and treatment arm. Two-dimensional (2D) echocardiographywas performed on test animals using a Vivid 7 Dimension ultrasound (GEHealthcare) with a 14-MHz transducer. To observe possible structuralremodeling, left ventricular end-diastolic diameter and free wallend-diastolic thickness were measured during diastole from long andshort-axis images obtained with M-mode echocardiography. Systolicfunction was assessed by the ejection fraction, and diastolic functionwas determined by taking trans-mitral flow measurements of ventricularfilling velocity using pulsed Doppler in an apical four-chamberorientation to determine the E/A ratio, isovolumetric relaxation time,and the E wave deceleration time, indicators of diastolic dysfunctionexplained further below.

The E/A ratio is the ratio of the peak velocity of blood movement fromthe left atrium to the left ventricle during two stages of atrialemptying and ventricular filling. Blood is transferred from the leftatrium to the left ventricle in two steps. In the first, the blood inthe left atrium moves passively into the ventricle below when the mitralvalve opens due to negative pressure created by the relaxing ventricle.The speed at which the blood moves during this initial action is calledthe “E,” for early, ventricular filling velocity. Later in time, theleft atrium contracts to eject any remaining blood in the atrium, andthe speed at which the blood moves at this stage is called the “A,” foratrium, ventricular filling velocity. The E/A ratio is the ratio of theearly (E) to late (A) ventricular filling velocities. In healthy heart,the E/A ratio is greater than 1. In Duchenne myopathy, however, the leftventricular wall becomes stiff, reducing ventricular relaxation and pullon atrial blood, thereby slowing the early (E) filling velocity andlowering the E/A ratio. The isovolumetric relaxation time (IVRT) is theinterval between the closure of the aortic valve to onset of ventricularfilling by opening of the mitral valve, or the time until ventricularfilling starts after relaxation begins. Longer than normal IVRTindicates poor ventricular relaxation, which has been described in bothhuman DMD patients (RC Bahler et al., J Am Soc Echocardiogr 18(6),666-73 (2005); L W Markham et al., J Am Soc Echocardiogr 19(7), 865-71(2006)) and the DMD dog model (V Chetboul et al., Eur Heart J 25(21),1934-39 (2004); V Chetboul et al., Am J Vet Res 65(10), 1335-41 (2004)),and precede the dilated cardiomyopathy associated with DMD. Lastly, theE wave deceleration time (DT) corresponds to the time in millisecondsbetween peak E velocity and its return to baseline, an increase in whichis indicative of a diastolic dysfunction.

Results

At both 3 and 6 months post-injection, no significant differences infree wall diastolic thickness between WT rats and Dmd^(mdx) rats, bothtreated with vehicle, indicating that this measurement was notinformative regarding disease course in this model at the ages examined.At 6 months, but not 3 months, post-injection, however, there was atrend toward increasing left ventricular end-diastolic diameter inDmd^(mdx) rats treated with vehicle compared to WT controls, which wasreversed when the Dmd^(mdx) rats were treated with vector, althoughstatistical significance was not reached (FIG. 43 ).

To assess systolic function, left ventricular (LV) ejection fraction wasmeasured. No difference was found in Dmd^(mdx) rats 3 monthspost-injection, but at 6 months post-injection, Dmd^(mdx) ratsadministered vehicle only exhibited reduced LV ejection fraction thatwas prevented by treatment with vector, although the difference wasstatistically significant only at one of the lower doses, 3×10¹³ vg/kg(FIG. 44 ).

To assess diastolic dysfunction, Doppler echocardiography was used tomeasure early (E) and late diastolic (A) velocities, the E/A ratio,isovolumetric relaxation time (IVRT), and deceleration time (DT). At 3months post-injection there was a statistically significant reduction inthe E/A ratio for Dmd^(mdx) rats treated with vehicle compared to WTcontrols, and a trend suggesting return to a normal E/A ratio inDmd^(mdx) rats treated with the highest vector dose, 3×10¹⁴ vg/kg,although the difference did not reach statistical significance (FIG.45A). At 6 months post-injection, the E/A ratio of Dmd^(mdx) ratstreated with vehicle were also significantly reduced compared to WTcontrols, and as with the earlier time point, there was a trendsuggesting some treatment effect of the vector, although the data wasquite variable and did not reach statistical significance (FIG. 45B).

At 3 months post-injection, IVRT was elevated in Dmd^(mdx) rats treatedwith vehicle compared to WT controls, and there was a slight trendsuggesting a dose responsive reduction in IVRT in Dmd^(mdx) rats treatedwith vector, although none of the differences in the data reachedstatistical significance (FIG. 46A). At 6 months post-injection,Dmd^(mdx) rats treated with vehicle had an IVRT that was significantlyhigher compared to WT controls, whereas vector treatment resulted in astrong trend suggesting return of IVRT to normal levels, which reachedstatistical significance at the lowest vector dose, 1×10¹³ vg/kg (FIG.46B).

Finally, DT could only be measured in older rats due to technicaldifficulties with an anesthesia protocol. When examined at 6 monthspost-injection, however, DT was significantly elevated in Dmd^(mdx) ratstreated with vehicle compared to WT controls, and there was a strongtrend toward restoration to normal values after vector treatment at alldoses tested (FIG. 47 ).

Despite variability in the data, the results of these studies stronglysuggest the existence of diastolic dysfunction in the hearts of 5 and 8month old Dmd^(mdx) rats, which could be at least partially reversed bytreatment with AAV9.hCK.Hopti-Dys3978.spA vector.

Blood Chemistry

Prior to treatment and at the time of sacrifice, blood samples from therats were taken and stored for eventual analysis. Tests were carried outto determine serum concentrations of urea, creatinine, alkalinephosphatase (ALK), alanine aminotransferase (ALT), aspartateaminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase(CK), troponin I, and antibodies against the mini-dystrophin protein andAAV9 capsid. ALT, AST, CK, and LDH are all enzymes released into theblood from damaged muscle cells, and are known to be elevated in humanDMD patients.

At 3 months and 6 months post-injection, the levels of urea, creatinine,ALK, total serum proteins, total bilirubin and troponin I were notsignificantly different between the different experimental groups. Bycontrast, AST, ALT, LDH and total CK levels were all elevated in vehicletreated Dmd^(mdx) rats compared to WT rats and responded with varyingdegrees to vector treatment.

At both 3 and 6 months post-injection, AST levels were elevated inDmd^(mdx) rats treated with vehicle compared to WT rats, although due tovariability in the data, significance existed only at the 6 month timepoint. When Dmd^(mdx) rats were treated with vector, a trend towardslower AST levels (albeit with wide inter-individual variability) wasobserved in the 1×10¹⁴ and 3×10¹⁴ vg/kg dose groups at 3 monthspost-injection and in the 3×10¹⁴ vg/kg dose group at 6 monthspost-injection. Again, due to variability in the data, these differencesdid not reach statistical significance. These results are shown in FIG.48A and FIG. 48B, which reports data for the 3 month and 6 monthpost-injection time points, respectively.

The pattern of ALT, LDH, and total CK levels all responded to age andvector treatment in similar ways. At 3 months post-injection, ALT, LDHand total CK levels were all significantly elevated in Dmd^(mdx) ratstreated with vehicle compared to WT rats. Treating the Dmd^(mdx) ratswith the mini-dystrophin vector resulted in a trend suggesting a doseresponsive reduction in ALT, LDH and total CK levels relative to vehicletreated Dmd^(mdx) rats, which in some cases achieved statisticalsignificance. These results are shown in FIG. 49A, FIG. 50A, and FIG.51A, respectively. At 6 months post-injection, there was a trend in thedata suggesting elevated levels of ALT and LDH in Dmd^(mdx) rats treatedwith vehicle compared to WT rats, which was reversed at highest vectordose tested, but none of the differences were statistically significant.These results are shown in FIG. 49B and FIG. 50B, respectively. Incontrast, similar to the pattern seen at 3 months post-injection, totalCK was significantly elevated in Dmd^(mdx) rats treated with vehicle at6 months post-injection compared to WT rats, and vector treatmentresulted in a trend toward reduced levels that achieved statisticalsignificance at the highest vector dose tested (FIG. 51B).

Total CK levels within treatment arms were also compared on the day ofinjection and 3 and 6 months after. As shown in FIG. 52A and FIG. 52B,blood total CK levels were consistently low in WT rats administeredvehicle, while CK levels declined in all Dmd^(mdx) rats, including thosetreated only with vehicle and the lowest vector dose. In contrast, thereduction of CK levels after 3 and 6 months was much greater forDmd^(mdx) rats treated with the three highest doses of vector. Theseobservations are consistent with the natural course of DMID in humans,where CK levels, while elevated compared to controls, decline as thedisease progresses due to replacement of muscle with adipose andfibrotic tissue, but also with a dose-responsive therapeutic effect atthe higher vector doses tested.

Differences in CK isoenzymes were also observed. Before dosing, theCK-MM isoform predominated in Dmd^(mdx) rats (mean >90%), whereas theCK-MM and CK-BB isoforms were comparable in WT rats (mean 40-60%), andCK-MB levels were higher in WT than in Dmd^(mdx) rats (4-6% versus ≈1%).At 3 and 6 months post-injection, Dmd^(mdx) rats treated with vectordoses above 1×10¹³ vg/kg showed a slight increase in the proportion ofthe CK-BB isoform and a slight decrease in the proportion of the CK-MMisoform, with a trend towards a dose-related effect. No clear alterationin the proportion of the CK-MB isoform was observed in vector treatedDmd^(mdx) rats.

Immunology

The humoral and cellular immune response in Dmd^(mdx) rats treated withAAV9.hCK.Hopti-Dys3978.spA vector were measured before treatment and at3 and 6 months post-injection and compared to negative and positivecontrols. Serum samples were obtained before injection of vehicle orvector, and at euthanasia 3 months post-injection. Splenocytes foranalysis of T cell response were harvested at euthanasia at 3 and 6months post-injection.

Humoral response to expression of the mini-dystrophin protein wasassessed qualitatively by Western blot analysis of sera obtained fromthe test animals and diluted 1:500. Sera from all rats, whether WT orDmd^(mdx), were negative for antibodies against mini-dystrophin proteinwhen administered vehicle, or prior to receiving vector. By contrastmost Dmd^(mdx) rats treated with vector, even at the lowest dose of1×10¹³ vg/kg, produced IgG antibodies that bound mini-dystrophin inWestern blots. Between 80%-100% of Dmd^(mdx) rats sacrificed at 3 monthspost-injection, and between 60%-100% of Dmd^(mdx) rats sacrificed at 6months post-injection produced IgG specific for the mini-dystrophinprotein depending on dose.

Presence of antibodies to the AAV9 vector capsid was tested by ELISA.Serum from WT and Dmd^(mdx) rats treated with vehicle had no detectableIgG that reacted with AAV9. By contrast, all rats treated with vector,regardless of dose or whether sacrificed 3 or 6 months post-injection,produced anti-AAV9 IgG with a titer higher than 1:10240, the highestdilution tested. Neutralizing antibodies against AAV9 were also testedwith a cell transduction inhibition assay using a recombinant AAV9vector that expresses LacZ reporter gene detected using a luminometer.The titer was defined as the lowest dilution that inhibitedtransduction >50%. Neutralizing antibodies against AAV9 were detected inthe serum from all Dmd^(mdx) rats that had received vector, regardlessof dose or whether sacrificed 3 or 6 months post-injection, but not inthe same animals prior to injection or WT and Dmd^(mdx) rats that hadreceived vehicle only. Titers ranged from 1:5000 to ≥1:500000 with noclear dose effect.

Presence of a cellular immune response to vector was evaluated using anIFNγ ELISpot assay on splenocytes isolated from vehicle treated WT andDmd^(mdx) rats, and Dmd^(mdx) rats that had received vector. T cellresponse to the human mini-dystrophin protein expressed by the vectorgenome was tested using an overlapping peptide bank covering the wholesequence of opti-dys3978 protein (length of 15 amino acids, overlap of10 amino acids, total of 263 peptides) and a rat specificIFNγ-ELISpot^(BASIC) kit (Mabtech). Negative control consisted ofunstimulated splenocytes and positive control consisted of cellsstimulated with the mitogen concanavalin A. IFNγ secretion wasquantified as the number of spot-forming cells (SFC) per 10⁶ cells, anda positive response was defined as >50 SFC/10⁶ cells or at least 3-foldthe value obtained for the negative control. No specific T cell responseagainst any peptide sequences derived from the mini-dystrophin proteinwas found in splenocytes obtained from any of the test animals, ateither 3 months or 6 months post-injection, including from Dmd^(mdx)rats treated at the highest vector dose of 3×10¹⁴ vg/kg.

T cell response against the AAV9 capsid was also tested using the IFNγELISpot assay screened against peptide sequences derived from AAV9(15-mers overlapping by 10 amino acids divided into 3 pools). There wasa positive IFNγ response in between 16%-60% of vector treated Dmd^(mdx)rats sacrificed at 3 months post-injection, and between 16%-66% ofvector treated Dmd^(mdx) rats sacrificed at 6 months post-injection,that was positively correlated with vector dose. By contrast, all WT andDmd^(mdx) rats treated with vehicle were negative for T cell responseagainst AAV9 capsid.

Example 9 Grip Strength in Older Dmd^(mdx) Rats Treated withAAV9.hCK.Hopti-Dys3978.spA

The studies described in Example 8, above, were initiated in young rats7-9 weeks of age. This example describes muscle function analysis ofolder Dmd^(mdx) rats first treated with the AAV9.hCK.Hopti-Dys3978.spAvector when they were 4 months of age and 6 months of age, respectively.The average life span of Sprague Dawley rats is 24-36 months. The goalof these experiments was to determine if treatment with vector later ina Dmd^(mdx) rat's life might be effective. Positive results wouldsuggest that treating older human DMD patients, such as older children,adolescents, or even young adults, with vector might also improve theirmuscle function.

The experiments described in this example were conducted using similarmaterials and methods as those described in Example 8. Morespecifically, Dmd^(mdx) rats at 4 and 6 months of age (n=6 for each agegroup) were separately treated with 1×10¹⁴ vg/kg ofAAV9.hCK.Hopti-Dys3978.spA vector. As negative controls, WT rats andDmd^(mdx) rats (n=6 for each age group) 4 months and 6 months of agewere separately treated with vehicle only. At 3 months post-injection,rats were tested for grip strength as described previously. As with theyounger rats, maximum forelimb grip strength and grip strength overmultiple repeated trials with short latency periods between each trialwere tested. The latter test was intended to measure muscle fatigue.

As shown in FIG. 53A, at 3 months post-injection, maximum forelimb gripstrength of Dmd^(mdx) rats treated with vehicle at 4 months of age wason average slightly lower compared to 4 month old WT rats treated withvehicle, although the difference did not reach statistical significance.By contrast, Dmd^(mdx) rats injected with 1×10¹⁴ vg/kg vector at 4months of age had greater average maximum forelimb grip strength thanDmd^(mdx) rats treated only with vehicle at the same age, a differencethat did reach statistical significance. The strength of the vectortreated rats was even greater than WT rats, although that difference wasnot statistically significant. The results were similar when the datawas normalized for body weight, as shown in FIG. 53B. In FIG. 53A andFIG. 53B, the symbol “

” indicates a statistically significant difference between vector versusvehicle treated Dmd^(mdx) rats (p<0.01).

With respect to muscle fatigue, as shown in FIG. 53C, Dmd^(mdx) ratstreated with vehicle at 4 months exhibited fatigue after the 2nd griptest, whereas WT rats exhibited no fatigue even after 4 tests. Bycontrast, 4 month old Dmd^(mdx) rats treated with vector exhibitedminimal, if any, muscle fatigue between the 1st and 5th grip tests. Thevector treated Dmd^(mdx) rats also appeared stronger overall compared toWT rats treated with vehicle. In FIG. 53C, the symbol “*” indicates astatistically significant difference between vector treated Dmd^(mdx)rats and WT rats treated with vehicle (p<0.05); “

” indicates a statistically significant difference between vector versusvehicle treated Dmd^(mdx) rats (p<0.01); and “§§ ” and “§§§ ” indicate astatistically significant difference between vehicle treated Dmd^(mdx)rats at the 4th and 5th grip tests, respectively, compared to the 1stgrip test (at p<0.01 and p<0.001, respectively).

As shown in FIG. 54A, at 3 months post-injection, maximum forelimb gripstrength of Dmd^(mdx) rats treated with vehicle at 6 months of age wassignificantly lower compared to 6 month old WT rats treated withvehicle. This effect was maintained even when the results werenormalized for body weight, as shown in FIG. 54B. Treating Dmd^(mdx)rats with 1×10¹⁴ vg/kg vector at 6 months of age increased the averagemaximum forelimb grip strength compared with Dmd^(mdx) rats treated onlywith vehicle, a difference that reached statistical significance whennormalized for body weight (FIG. 54B). In FIG. 54A and FIG. 54B, thesymbols “*” and “**” indicate a statistically significant differencebetween vehicle treated Dmd^(mdx) and WT rats (at p<0.05 and p<0.01,respectively); and “

” indicates a statistically significant difference between vector versusvehicle treated Dmd^(mdx) rats (p<0.05).

With respect to muscle fatigue, as shown in FIG. 54C, Dmd^(mdx) ratstreated with vehicle at 6 months exhibited fatigue after the 2nd griptest, whereas WT rats exhibited no fatigue even after 4 tests. Incontrast with rats treated at 4 months, 6 month old Dmd^(mdx) ratstreated with vector exhibited some reduced strength over the multiplegrip tests, although not to the same extent as that seen with vehicletreated control Dmd^(mdx) rats. Also in contrast to the tests conductedwith rats treated at 4 months of age, the strength of the WT ratsappeared to be greater than that of Dmd^(mdx) rats treated with vectorat 6 months over the course of the experiment. In FIG. 54C, the symbols“**” and “***” indicate a statistically significant difference betweenvector treated Dmd^(mdx) rats and WT rats treated with vehicle (atp<0.01 and p<0.001, respectively); “

” indicates a statistically significant difference between vector versusvehicle treated Dmd^(mdx) rats (p<0.05); and “§§ ” indicates astatistically significant difference between vehicle treated Dmd^(mdx)rats at the 5th grip test compared to the 1st grip test (p<0.01).

TABLE 13 TABLE QF SEQUENCES SEQ ID DESCRIPTION AND NO SEQUENCE  SEQ IDDNA sequence of human codon-optimized gene encoding human mini-dystrophin 3978 (Hopti-Dys3978)NO: 1 atgctttggt gggaggaagt ggaggactgc tacgagagag aggacgtgca gaagaaaacc60 ttcaccaagt gggtgaacgc ccagttcagc aagttcggca agcagcacat cgagaacctg 120ttcagcgacc tgcaggatgg caggagactg ctggacctgc tggagggcct gaccggccag 180aagctgccca aggagaaggg cagcaccaga gtgcacgccc tgaacaacgt gaacaaggcc 240ctgagagtgc tgcagaacaa caacgtggac ctggtgaaca tcggcagcac cgacatcgtg 300gacggcaacc acaagctgac cctgggcctg atctggaaca tcatcctgca ctggcaggtg 360aagaacgtga tgaagaacat catggccggc ctgcagcaga ccaacagcga gaagatcctg 420ctgagctggg tgaggcagag caccagaaac tacccccagg tgaacgtgat caacttcacc 480acctcctgga gcgacggcct ggccctgaac gccctgatcc acagccacag acccgacctg 540ttcgactgga acagcgtggt gtgtcagcag agcgccaccc agagactgga gcacgccttc 600aacatcgcca gataccagct gggcatcgag aagctgctgg accccgagga cgtggacacc 660acctaccccg acaagaaaag catcctcatg tacattacca gcctgttcca ggtgctgccc 720cagcaggtgt ccatcgaggc catccaggaa gtggaaatgc tgcccaggcc ccccaaagtg 780accaaggagg agcacttcca gctgcaccac cagatgcact acagccagca gatcacagtg 840agcctggccc agggctatga gagaaccagc agccccaagc ccagattcaa gagctacgcc 900tacacccagg ccgcctacgt gaccacctcc gaccccacca gaagcccctt ccccagccag 960cacctggagg cccccgagga caagagcttc ggcagcagcc tgatggagag cgaagtgaac 1020ctggacagat accagaccgc cctggaggaa gtgctgtcct ggctgctgag cgccgaggac 1080accctgcagg cccagggcga gatcagcaac gacgtggaag tggtgaagga ccagttccac 1140acccacgagg gctacatgat ggatctgacc gcccaccagg gcagagtggg caatatcctg 1200cagctgggca gcaagctgat cggcaccggc aagctgagcg aggacgagga gaccgaagtg 1260caggagcaga tgaacctgct gaacagcaga tgggagtgcc tgagagtggc cagcatggag 1320aagcagagca acctgcacag agtgctgatg gacctgcaga accagaagct gaaggagctg 1380aacgactggc tgaccaagac cgaggagcgg accagaaaga tggaggagga gcccctgggc 1440cccgacctgg aggacctgaa gagacaggtg cagcagcaca aagtgctgca ggaggacctg 1500gagcaggagc aggtgcgcgt gaacagcctg acccacatgg tggtggtcgt ggacgagagc 1560agcggcgacc acgccacagc cgccctggaa gagcagctga aagtgctggg cgacagatgg 1620gccaatattt gtaggtggac cgaggacaga tgggtgctgc tgcaggacca gcccgacctg 1680gcccctggcc tgaccaccat cggcgccagc cccacccaga ccgtgaccct ggtgacccag 1740cccgtggtga caaaggagac cgccatcagc aagctggaga tgcccagctc cctgatgctg 1800gaagtgccca cccaccgcct gctccagcag ttccccctgg acctggagaa gttcctggcc 1860tggctgaccg aggccgaaac caccgccaat gtgctccagg acgccactag aaaggagagg 1920ctgctggagg acagcaaggg cgtgaaagag ctgatgaagc agtggcagga tctgcagggc 1980gaaatcgagg cccacaccga cgtgtaccac aacctggacg agaacagcca gaagattctg 2040aggagcctgg agggcagcga cgacgccgtc ctgctccaga ggaggctgga caacatgaac 2100ttcaagtgga gcgagctgcg gaagaagagc ctgaacatcc ggagccacct ggaagccagc 2160agcgaccagt ggaagagact gcacctgagc ctgcaggagc tgctggtgtg gctgcagctg 2220aaggacgacg agctgagcag acaggccccc atcggcggcg acttccccgc cgtgcagaag 2280cagaacgacg tgcaccgggc cttcaagagg gagctgaaaa ccaaggaacc cgtgatcatg 2340agcaccctgg agacagtgcg gatcttcctg accgagcagc ccctggaggg actggagaag 2400ctgtaccagg agcccagaga gctgcccccc gaggagagag cccagaacgt gaccaggctg 2460ctgagaaagc aggccgagga agtgaatacc gagtgggaga agctgaatct gcacagcgcc 2520gactggcaga gaaagatcga cgagaccctg gagagactcc aggaactgca ggaagccacc 2580gacgagctgg acctgaagct gagacaggcc gaagtgatca agggcagctg gcagcctgtg 2640ggcgatctgc tgatcgactc cctgcaggat cacctggaga aagtgaaggc cctgcggggc 2700gagatcgccc ccctgaagga gaatgtgagc cacgtgaacg acctggccag acagctgacc 2760accctgggca tccagctgag cccctacaac ctgagcacac tggaggatct gaacacccgg 2820tggaaactgc tgcaggtggc cgtggaggat agagtgaggc agctgcacga agcccacaga 2880gacttcggcc ctgcctccca gcacttcctg agcaccagcg tgcagggccc ctgggagaga 2940gccatctccc ccaacaaagt gccctactac atcaaccacg agacccagac cacctgctgg 3000gaccacccta agatgaccga gctgtatcag agcctggccg acctgaacaa tgtgcggttc 3060agcgcctaca gaaccgccat gaagctgcgg agactgcaga aggccctgtg cctggatctg 3120ctgagcctga gcgccgcctg cgacgccctg gaccagcaca acctgaagca gaatgaccag 3180cccatggaca tcctgcagat catcaactgc ctgaccacaa tctacgaccg gctggaacag 3240gagcacaaca acctggtgaa tgtgcccctg tgcgtggaca tgtgcctgaa ttggctgctg 3300aacgtgtacg acaccggcag gaccggcaga atccgcgtgc tgagcttcaa gaccggcatc 3360atcagcctgt gcaaggccca cctggaggat aagtaccgct acctgttcaa gcaggtggcc 3420agcagcaccg gcttctgcga tcagaggaga ctgggcctgc tgctgcacga tagcatccag 3480atccctaggc agctgggcga agtggccagc tttggcggca gcaacatcga gccctctgtg 3540aggagctgct tccagttcgc caacaacaag cccgagatcg aggccgccct gttcctggac 3600tggatgaggc tggagcctca gagcatggtg tggctgcctg tgctgcacag agtggccgcc 3660gccgagaccg ccaagcacca ggccaagtgc aatatctgca aggagtgccc catcatcggc 3720ttccggtaca ggagcctgaa gcacttcaac tacgacatct gccagagctg ctttttcagc 3780ggcagagtgg ccaagggcca caaaatgcac taccccatgg tggagtactg cacccccacc 3840acctccggcg aggatgtgag agacttcgcc aaagtgctga agaataagtt ccggaccaag 3900cggtactttg ccaagcaccc caggatgggc tacctgcccg tgcagaccgt gctggaaggc 3960gacaacatgg agacctga 3978 SEQ IDDNA sequence of human codon-optimized gene encoding human mini-dystrophin 3837 (Hopti-Dys3837)NO: 2 atgctttggt gggaggaagt ggaggactgc tacgagagag aggacgtgca gaagaaaacc60 ttcaccaagt gggtgaacgc ccagttcagc aagttcggca agcagcacat cgagaacctg 120ttcagcgacc tgcaggatgg caggagactg ctggacctgc tggagggcct gaccggccag 180aagctgccca aggagaaggg cagcaccaga gtgcacgccc tgaacaacgt gaacaaggcc 240ctgagagtgc tgcagaacaa caacgtggac ctggtgaaca tcggcagcac cgacatcgtg 300gacggcaacc acaagctgac cctgggcctg atctggaaca tcatcctgca ctggcaggtg 360aagaacgtga tgaagaacat catggccggc ctgcagcaga ccaacagcga gaagatcctg 420ctgagctggg tgaggcagag caccagaaac tacccccagg tgaacgtgat caacttcacc 480acctcctgga gcgacggcct ggccctgaac gccctgatcc acagccacag acccgacctg 540ttcgactgga acagcgtggt gtgtcagcag agcgccaccc agagactgga gcacgccttc 600aacatcgcca gataccagct gggcatcgag aagctgctgg accccgagga cgtggacacc 660acctaccccg acaagaaaag catcctcatg tacattacca gcctgttcca ggtgctgccc 720cagcaggtgt ccatcgaggc catccaggaa gtggaaatgc tgcccaggcc ccccaaagtg 780accaaggagg agcacttcca gctgcaccac cagatgcact acagccagca gatcacagtg 840agcctggccc agggctatga gagaaccagc agccccaagc ccagattcaa gagctacgcc 900tacacccagg ccgcctacgt gaccacctcc gaccccacca gaagcccctt ccccagccag 960cacctggagg cccccgagga caagagcttc ggcagcagcc tgatggagag cgaagtgaac 1020ctggacagat accagaccgc cctggaggaa gtgctgtcct ggctgctgag cgccgaggac 1080accctgcagg cccagggcga gatcagcaac gacgtggaag tggtgaagga ccagttccac 1140acccacgagg gctacatgat ggatctgacc gcccaccagg gcagagtggg caatatcctg 1200cagctgggca gcaagctgat cggcaccggc aagctgagcg aggacgagga gaccgaagtg 1260caggagcaga tgaacctgct gaacagcaga tgggagtgcc tgagagtggc cagcatggag 1320aagcagagca acctgcacag agtgctgatg gacctgcaga accagaagct gaaggagctg 1380aacgactggc tgaccaagac cgaggagcgg accagaaaga tggaggagga gcccctgggc 1440cccgacctgg aggacctgaa gagacaggtg cagcagcaca aagtgctgca ggaggacctg 1500gagcaggagc aggtgcgcgt gaacagcctg acccacatgg tggtggtcgt ggacgagagc 1560agcggcgacc acgccacagc cgccctggaa gagcagctga aagtgctggg cgacagatgg 1620gccaatattt gtaggtggac cgaggacaga tgggtgctgc tgcaggacac ccaccgcctg 1680ctccagcagt tccccctgga cctggagaag ttcctggcct ggctgaccga ggccgaaacc 1740accgccaatg tgctccagga cgccactaga aaggagaggc tgctggagga cagcaagggc 1800gtgaaagagc tgatgaagca gtggcaggat ctgcagggcg aaatcgaggc ccacaccgac 1860gtgtaccaca acctggacga gaacagccag aagattctga ggagcctgga gggcagcgac 1920gacgccgtcc tgctccagag gaggctggac aacatgaact tcaagtggag cgagctgcgg 1980aagaagagcc tgaacatccg gagccacctg gaagccagca gcgaccagtg gaagagactg 2040cacctgagcc tgcaggagct gctggtgtgg ctgcagctga aggacgacga gctgagcaga 2100caggccccca tcggcggcga cttccccgcc gtgcagaagc agaacgacgt gcaccgggcc 2160ttcaagaggg agctgaaaac caaggaaccc gtgatcatga gcaccctgga gacagtgcgg 2220atcttcctga ccgagcagcc cctggaggga ctggagaagc tgtaccagga gcccagagag 2280ctgccccccg aggagagagc ccagaacgtg accaggctgc tgagaaagca ggccgaggaa 2340gtgaataccg agtgggagaa gctgaatctg cacagcgccg actggcagag aaagatcgac 2400gagaccctgg agagactcca ggaactgcag gaagccaccg acgagctgga cctgaagctg 2460agacaggccg aagtgatcaa gggcagctgg cagcctgtgg gcgatctgct gatcgactcc 2520ctgcaggatc acctggagaa agtgaaggcc ctgcggggcg agatcgcccc cctgaaggag 2580aatgtgagcc acgtgaacga cctggccaga cagctgacca ccctgggcat ccagctgagc 2640ccctacaacc tgagcacact ggaggatctg aacacccggt ggaaactgct gcaggtggcc 2700gtggaggata gagtgaggca gctgcacgaa gcccacagag acttcggccc tgcctcccag 2760cacttcctga gcaccagcgt gcagggcccc tgggagagag ccatctcccc caacaaagtg 2820ccctactaca tcaaccacga gacccagacc acctgctggg accaccctaa gatgaccgag 2880ctgtatcaga gcctggccga cctgaacaat gtgcggttca gcgcctacag aaccgccatg 2940aagctgcgga gactgcagaa ggccctgtgc ctggatctgc tgagcctgag cgccgcctgc 3000gacgccctgg accagcacaa cctgaagcag aatgaccagc ccatggacat cctgcagatc 3060atcaactgcc tgaccacaat ctacgaccgg ctggaacagg agcacaacaa cctggtgaat 3120gtgcccctgt gcgtggacat gtgcctgaat tggctgctga acgtgtacga caccggcagg 3180accggcagaa tccgcgtgct gagcttcaag accggcatca tcagcctgtg caaggcccac 3240ctggaggata agtaccgcta cctgttcaag caggtggcca gcagcaccgg cttctgcgat 3300cagaggagac tgggcctgct gctgcacgat agcatccaga tccctaggca gctgggcgaa 3360gtggccagct ttggcggcag caacatcgag ccctctgtga ggagctgctt ccagttcgcc 3420aacaacaagc ccgagatcga ggccgccctg ttcctggact ggatgaggct ggagcctcag 3480agcatggtgt ggctgcctgt gctgcacaga gtggccgccg ccgagaccgc caagcaccag 3540gccaagtgca atatctgcaa ggagtgcccc atcatcggct tccggtacag gagcctgaag 3600cacttcaact acgacatctg ccagagctgc tttttcagcg gcagagtggc caagggccac 3660aaaatgcact accccatggt ggagtactgc acccccacca cctccggcga ggatgtgaga 3720gacttcgcca aagtgctgaa gaataagttc cggaccaagc ggtactttgc caagcacccc 3780aggatgggct acctgcccgt gcagaccgtg ctggaaggcg acaacatgga gacctga 3837SEQ IDDNA sequence of canine codon-optimized gene encoding human mini-dystrophin 3978 (Copti-Dys3978)NO: 3 atgctttggt gggaggaagt ggaggactgc tacgagcggg aggacgtgca gaagaaaacc60 ttcaccaagt gggtgaacgc ccagttcagc aagttcggca agcagcacat cgagaacctg 120ttcagcgacc tgcaggacgg caggcggctg ctggacctcc tggaaggcct gaccggccag 180aagctgccca aagagaaggg cagcaccagg gtgcacgccc tgaacaacgt gaacaaggcc 240ctgagggtgc tgcagaacaa caacgtggac ctggtgaaca tcggcagcac cgacatcgtg 300gacggcaacc acaagctgac cctgggcctg atctggaaca tcatcctgca ctggcaggtc 360aagaacgtga tgaagaacat catggccggc ctgcagcaga ccaacagcga gaagatcctg 420ctgtcctggg tgcggcagag caccaggaac tacccccagg tcaacgtgat caacttcacc 480acctcttgga gcgacggcct ggccctgaac gccctgatcc acagccacag gcccgacctg 540ttcgactgga acagcgtggt gtgccagcag agcgccaccc agaggctgga acacgccttc 600aacatcgcca gataccagct gggcatcgag aagctgctgg atcccgagga cgtggacacc 660acctaccccg acaagaaaag catcctcatg tacatcacca gcctgttcca ggtgctgccc 720cagcaggtgt ccatcgaggc catccaggaa gtggagatgc tgcccaggcc ccccaaggtc 780accaaagagg aacacttcca gctgcaccac cagatgcact acagccagca gatcaccgtg 840agcctggccc agggctacga gaggaccagc agccccaagc ccaggttcaa gagctacgcc 900tacacccagg ccgcctacgt gaccacctcc gaccccacca ggtccccctt ccccagccag 960catctcgaag cccccgagga caagagcttc ggcagcagcc tgatggaaag cgaggtgaac 1020ctggacagat accagaccgc cctggaagaa gtgctgtctt ggctgctgtc cgccgaggac 1080accctgcagg cccagggcga gatcagcaac gacgtggagg tcgtgaagga ccagttccac 1140acccacgagg gctacatgat ggacctgacc gcccaccagg gcagagtggg caacatcctg 1200cagctgggca gcaagctgat cggcaccggc aagctgtccg aggacgagga aaccgaggtg 1260caggaacaga tgaacctgct gaacagcaga tgggagtgcc tgagggtggc cagcatggaa 1320aagcagagca acctgcacag ggtgctgatg gatctgcaga accagaagct caaagagctg 1380aacgactggc tgaccaagac cgaggaaagg acccggaaga tggaagagga acccctgggccccgatctcg aagatctgaa gaggcaggtg cagcagcaca aggtgctgca ggaagatctc 1500gaacaggaac aggtccgggt caacagcctg acccacatgg tcgtggtggt ggacgagagc 1560agcggcgacc acgccaccgc tgccctggaa gagcagctga aggtgctggg cgacagatgg 1620gccaacatct gccggtggac cgaggacaga tgggtcctcc tgcaggacca gcccgacctg 1680gcccctggcc tgacaaccat cggcgccagc cccacccaga ccgtgaccct ggtgacccag 1740cccgtggtga ccaaagagac cgccatcagc aagctggaaa tgcccagctc cctgatgctg 1800gaagtgccca cccacaggct cctccagcag ttccccctgg acctggaaaa gttcctggcc 1860tggctgaccg aggccgagac caccgccaac gtgctgcagg acgccaccag gaaagagagg 1920ctgctggaag atagcaaggg cgtgaaagag ctgatgaagc agtggcagga cctgcagggg 1980gagattgagg cccacaccga cgtgtaccac aacctggacg agaacagcca gaaaatcctg 2040agaagcctgg aaggcagcga cgacgccgtg ctgctgcaga ggcggctgga caacatgaac 2100ttcaagtgga gcgagctgag gaagaagagc ctgaacatca ggtcccatct ggaagccagc 2160agcgaccagt ggaagaggct gcacctgagc ctgcaggaac tgctcgtctg gctgcagctg 2220aaagacgacg agctgtccag gcaggccccc atcggcggcg acttccccgc cgtgcagaaa 2280cagaacgacg tgcacagggc cttcaagcgg gagctgaaaa ccaaagagcc cgtgatcatg 2340agcaccctgg aaaccgtgag gatcttcctg accgagcagc ccctggaagg actggaaaag 2400ctgtaccagg aacccagaga gctgcccccc gaggaacggg cccagaacgt gaccaggctg 2460ctgagaaagc aggccgagga agtgaacacc gagtgggaga agctgaacct gcactccgcc 2520gactggcaga ggaagatcga cgagaccctg gaaaggctcc aggaactgca ggaagccacc 2580gacgagctgg acctgaagct gagacaggcc gaggtgatca agggcagctg gcagcccgtg 2640ggcgacctgc tgatcgactc cctgcaggat cacctggaaa aagtgaaggc cctgcggggc 2700gagatcgccc ccctgaaaga gaacgtcagc cacgtcaacg acctggccag gcagctgacc 2760accctgggca tccagctgtc cccctacaac ctgtccaccc tggaagatct gaacacaagg 2820tggaagctgc tgcaggtggc cgtggaggac agagtgaggc agctgcacga ggcccacagg 2880gacttcggcc ctgcctccca gcacttcctg agcaccagcg tgcagggccc ctgggagagg 2940gccatctccc ccaacaaggt gccctactac atcaaccacg agacccagac cacctgctgg 3000gaccacccta agatgaccga gctgtaccag tccctggccg acctgaacaa tgtgcggttc 3060agcgcctacc ggaccgccat gaagctgagg cggctgcaga aagccctgtg cctggatctg 3120ctgtccctga gcgccgcctg cgacgccctg gaccagcaca acctgaagca gaacgaccag 3180cccatggata tcctgcagat catcaactgt ctgaccacca tctacgacag gctggaacag 3240gaacacaaca acctggtcaa cgtgcccctg tgcgtggaca tgtgcctgaa ctggctgctg 3300aacgtgtacg acaccggcag gaccggccgg atcagggtgc tgtccttcaa gaccggcatc 3360atcagcctgt gcaaggccca cctggaagat aagtaccgct acctgttcaa gcaggtggcc 3420agctctaccg gcttctgcga ccagcggagg ctgggcctgc tgctgcacga cagcatccag 3480atcccccggc agctgggcga ggtggcctcc ttcggcggca gcaacatcga gcccagcgtg 3540cggagctgct tccagttcgc caacaacaag cccgagatcg aggccgccct gttcctggac 3600tggatgcggc tggaacccca gagcatggtc tggctgcccg tgctgcacag agtggctgcc 3660gccgagaccg ccaagcacca ggccaagtgc aacatctgca aagagtgccc catcatcggc 3720ttcaggtaca gaagcctgaa gcacttcaac tacgacatct gccagagctg tttcttcagc 3780ggcagggtgg ccaagggcca caaaatgcac taccccatgg tggagtactg cacccccacc 3840acctccggcg aggacgtgag ggacttcgcc aaggtgctga agaataagtt ccggaccaag 3900cggtacttcg ccaaacaccc caggatgggc tacctgcccg tgcagaccgt gctggaaggc 3960gacaacatgg aaacctga 3978 SEQ IDDNA sequence of synthetic hybrid muscle-specific promoter hCK NO: 4gaattcggta ccccactacg ggtttaggct gcccatgtaa ggaggcaagg cctggggaca 60cccgagatgc ctggttataa ttaacccaga catgtggctg cccccccccc ccccaacacc 120tgctgcctct aaaaataacc ctgtccctgg tggatcccct gcatgcgaag atcttcgaac 180aaggctgtgg gggactgagg gcaggctgta acaggcttgg gggccagggc ttatacgtgc 240ctgggactcc caaagtatta ctgttccatg ttcccggcga agggccagct gtcccccgcc 300agctagactc agcacttagt ttaggaacca gtgagcaagt cagcccttgg ggcagcccat 360acaaggccat ggggctgggc aagctgcacg cctgggtccg gggtgggcac ggtgcccggg 420caacgagctg aaagctcatc tgctctcagg ggcccctccc tggggacagc ccctcctggc 480tagtcacacc ctgtaggctc ctctatataa cccaggggca caggggctgc cctcattcta 540ccaccacctc cacagcacag acagacactc aggagccagc cagcgtcgag cggccgatcc 600gccacc 606 SEQ IDDNA sequence of synthetic hybrid muscle-specific promoter hCKplus NO: 5gaattcggta ccccactacg ggtctaggct gcccatgtaa ggaggcaagg cctggggaca 60cccgagatgc ctggttataa ttaaccccaa cacctgctgc cccccccccc ccaacacctg 120ctgcctctaa aaataaccct gtccctggtg gatcccctgc atgccccact acgggtttag 180gctgcccatg taaggaggca aggcctgggg acacccgaga tgcctggtta taattaaccc 240agacatgtgg ctgccccccc cccccccaac acctgctgcc tctaaaaata accctgtccc 300tggtggatcc cctgcatgcg aagatcttcg aacaaggctg tgggggactg agggcaggct 360gtaacaggct tgggggccag ggcttatacg tgcctgggac tcccaaagta ttactgttcc 420atgttcccgg cgaagggcca gctgtccccc gccagctaga ctcagcactt agtttaggaa 480ccagtgagca agtcagccct tggggcagcc catacaaggc catggggctg ggcaagctgc 540acgcctgggt ccggggtggg cacggtgccc gggcaacgag ctgaaagctc atctgctctc 600aggggcccct ccctggggac agcccctcct ggctagtcac accctgtagg ctcctctata 660taacccaggg gcacaggggc tgccctcatt ctaccaccac ctccacagca cagacagaca 720ctcaggagcc agccagcgtc gagcggccga tccgccacc  759 SEQ IDDNA sequence of small synthetic polyadenylation element  NO: 6tgaggagctc gagaggccta ataaagagct cagatgcatc gatcagagtg tgttggtttt 60ttgtgtg 67 SEQ IDAmino acid sequence encoded by Hopti-Dys3978 gene (Dys3978 protein)NO: 7 MLWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ60 KLPKEKGSTR VHALNNVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV 120KNVMKNIMAG LQQTNSEKIL LSWVRQSTRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL 180FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP 240QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA 300YTQAAYVTTS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAED 360TLQAQGEISN DVEVVKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV 420QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG 480PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMVVVVDES SGDHATAALE EQLKVLGDRW 540ANICRWTEDR WVLLQDQPDL APGLTTIGAS PTQTVTLVTQ PVVTKETAIS KLEMPSSLML 600EVPTHRLLQQ FPLDLEKFLA WLTEAETTAN VLQDATRKER LLEDSKGVKE LMKQWQDLQG 660EIEAHTDVYH NLDENSQKIL RSLEGSDDAV LLQRRLDNMN FKWSELRKKS LNIRSHLEAS 720SDQWKRLHLS LQELLVWLQL KDDELSRQAP IGGDFPAVQK QNDVHRAFKR ELKTKEPVIM 780STLETVRIFL TEQPLEGLEK LYQEPRELPP EERAQNVTRL LRKQAEEVNT EWEKLNLHSA 840DWQRKIDETL ERLQELQEAT DELDLKLRQA EVIKGSWQPV GDLLIDSLQD HLEKVKALRG 900EIAPLKENVS HVNDLARQLT TLGIQLSPYN LSTLEDLNTR WKLLQVAVED RVRQLHEAHR 960DFGPASQHFL STSVQGPWER AISPNKVPYY INHETQTTCW DHPKMTELYQ SLADLNNVRF 1020SAYRTAMKLR RLQKALCLDL LSLSAACDAL DQHNLKQNDQ PMDILQIINC LTTIYDRLEQ 1080EHNNLVNVPL CVDMCLNWLL NVYDTGRTGR IRVLSFKTGI ISLCKAHLED KYRYLFKQVA 1140SSTGFCDQRR LGLLLHDSIQ IPRQLGEVAS FGGSNIEPSV RSCFQFANNK PEIEAALFLD 1200WMRLEPQSMV WLPVLHRVAA AETAKHQAKC NICKECPIIG FRYRSLKHFN YDICQSCFFS 1260GRVAKGHKMH YPMVEYCTPT TSGEDVRDFA KVLKNKFRTK RYFAKHPRMG YLPVQTVLEG 1320DNMET 1325 SEQ IDAmino acid sequence encoded by Hopti-Dys3837 gene (Dys3837 protein)NO: 8 MLWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ60 KLPKEKGSTR VHALNNVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV 120KNVMKNIMAG LQQTNSEKIL LSWVRQSTRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL 180FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP 240QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA 300YTQAAYVTTS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAED 360TLQAQGEISN DVEVVKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV 420QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG 480PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMVVVVDES SGDHATAALE EQLKVLGDRW 540ANICRWTEDR WVLLQDTHRL LQQFPLDLEK FLAWLTEAET TANVLQDATR KERLLEDSKG 600VKELMKQWQD LQGEIEAHTD VYHNLDENSQ KILRSLEGSD DAVLLQRRLD NMNFKWSELR 660KKSLNIRSHL EASSDQWKRL HLSLQELLVW LQLKDDELSR QAPIGGDFPA VQKQNDVHRA 720FKRELKTKEP VIMSTLETVR IFLTEQPLEG LEKLYQEPRE LPPEERAQNV TRLLRKQAEE 780VNTEWEKLNL HSADWQRKID ETLERLQELQ EATDELDLKL RQAEVIKGSW QPVGDLLIDS 840LQDHLEKVKA LRGEIAPLKE NVSHVNDLAR QLTTLGIQLS PYNLSTLEDL NTRWKLLQVA 900VEDRVRQLHE AHRDFGPASQ HELSTSVQGP WERAISPNKV PYYINHETQT TCWDHPKMTE 960LYQSLADLNN VRFSAYRTAM KLRRLQKALC LDLLSLSAAC DALDQHNLKQ NDQPMDILQI 1020INCLTTIYDR LEQEHNNLVN VPLCVDMCLN WLLNVYDTGR TGRIRVLSFK TGIISLCKAH 1080LEDKYRYLFK QVASSTGFCD QRRLGLLLHD SIQIPRQLGE VASFGGSNIE PSVRSCFQFA 1140NNKPEIEAAL FLDWMRLEPQ SMVWLPVLHR VAAAETAKHQ AKCNICKECP IIGFRYRSLK 1200HFNYDICQSC FFSGRVAKGH KMHYPMVEYC TPTTSGEDVR DFAKVLKNKF RTKRYFAKHP 1260RMGYLPVQTV LEGDNMET 1278 SEQ IDDNA sequence of a human-codon optimized human mini-dystrophin 3978 (Hopti-Dys3978) geneNO: 9 expression cassettettggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctagatc tgaattcggt accccactac gggtttaggc 180tgcccatgta aggaggcaag gcctggggac acccgagatg cctggttata attaacccag 240acatgtggct gccccccccc cccccaacac ctgctgcctc taaaaataac cctgtccctg 300gtggatcccc tgcatgcgaa gatcttcgaa caaggctgtg ggggactgag ggcaggctgt 360aacaggcttg ggggccaggg cttatacgtg cctgggactc ccaaagtatt actgttccat 420gttcccggcg aagggccagc tgtcccccgc cagctagact cagcacttag tttaggaacc 480agtgagcaag tcagcccttg gggcagccca tacaaggcca tggggctggg caagctgcac 540gcctgggtcc ggggtgggca cggtgcccgg gcaacgagct gaaagctcat ctgctctcag 600gggcccctcc ctggggacag cccctcctgg ctagtcacac cctgtaggct cctctatata 660acccaggggc acaggggctg ccctcattct accaccacct ccacagcaca gacagacact 720caggagccag ccagcgtcga gcggccgatc cgccaccatg ctttggtggg aggaagtgga 780ggactgctac gagagagagg acgtgcagaa gaaaaccttc accaagtggg tgaacgccca 840gttcagcaag ttcggcaagc agcacatcga gaacctgttc agcgacctgc aggatggcag 900gagactgctg gacctgctgg agggcctgac cggccagaag ctgcccaagg agaagggcag 960caccagagtg cacgccctga acaacgtgaa caaggccctg agagtgctgc agaacaacaa 1020cgtggacctg gtgaacatcg gcagcaccga catcgtggac ggcaaccaca agctgaccct 1080gggcctgatc tggaacatca tcctgcactg gcaggtgaag aacgtgatga agaacatcat 1140ggccggcctg cagcagacca acagcgagaa gatcctgctg agctgggtga ggcagagcac 1200cagaaactac ccccaggtga acgtgatcaa cttcaccacc tcctggagcg acggcctggc 1260cctgaacgcc ctgatccaca gccacagacc cgacctgttc gactggaaca gcgtggtgtg 1320tcagcagagc gccacccaga gactggagca cgccttcaac atcgccagat accagctggg 1380catcgagaag ctgctggacc ccgaggacgt ggacaccacc taccccgaca agaaaagcat 1440cctcatgtac attaccagcc tgttccaggt gctgccccag caggtgtcca tcgaggccat 1500ccaggaagtg gaaatgctgc ccaggccccc caaagtgacc aaggaggage acttccagct 1560gcaccaccag atgcactaca gccagcagat cacagtgagc ctggcccagg gctatgagag 1620aaccagcagc cccaagccca gattcaagag ctacgcctac acccaggccg cctacgtgac 1680cacctccgac cccaccagaa gccccttccc cagccagcac ctggaggccc ccgaggacaa 1740gagcttcggc agcagcctga tggagagcga agtgaacctg gacagatacc agaccgccct 1800ggaggaagtg ctgtcctggc tgctgagcgc cgaggacacc ctgcaggccc agggcgagat 1860cagcaacgac gtggaagtgg tgaaggacca gttccacacc cacgagggct acatgatgga 1920tctgaccgcc caccagggca gagtgggcaa tatcctgcag ctgggcagca agctgatcgg 1980caccggcaag ctgagcgagg acgaggagac cgaagtgcag gagcagatga acctgctgaa 2040cagcagatgg gagtgcctga gagtggccag catggagaag cagagcaacc tgcacagagt 2100gctgatggac ctgcagaacc agaagctgaa ggagctgaac gactggctga ccaagaccga 2160ggagcggacc agaaagatgg aggaggagcc cctgggcccc gacctggagg acctgaagag 2220acaggtgcag cagcacaaag tgctgcagga ggacctggag caggagcagg tgcgcgtgaa 2280cagcctgacc cacatggtgg tggtcgtgga cgagagcage ggcgaccacg ccacagccgc 2340cctggaagag cagctgaaag tgctgggcga cagatgggcc aatatttgta ggtggaccga 2400ggacagatgg gtgctgctgc aggaccagcc cgacctggcc cctggcctga ccaccatcgg 2460cgccagcccc acccagaccg tgaccctggt gacccagccc gtggtgacaa aggagaccgc 2520catcagcaag ctggagatgc ccagctccct gatgctggaa gtgcccaccc accgcctgct 2580ccagcagttc cccctggacc tggagaagtt cctggcctgg ctgaccgagg ccgaaaccac 2640cgccaatgtg ctccaggacg ccactagaaa ggagaggctg ctggaggaca gcaagggcgt 2700gaaagagctg atgaagcagt ggcaggatct gcagggcgaa atcgaggccc acaccgacgt 2760gtaccacaac ctggacgaga acagccagaa gattctgagg agcctggagg gcagcgacga 2820cgccgtcctg ctccagagga ggctggacaa catgaacttc aagtggagcg agctgcggaa 2880gaagagcctg aacatccgga gccacctgga agccagcagc gaccagtgga agagactgca 2940cctgagcctg caggagctgc tggtgtggct gcagctgaag gacgacgagc tgagcagaca 3000ggcccccatc ggcggcgact tccccgccgt gcagaagcag aacgacgtgc accgggcctt 3060caagagggag ctgaaaacca aggaacccgt gatcatgagc accctggaga cagtgcggat 3120cttcctgacc gagcagcccc tggagggact ggagaagctg taccaggagc ccagagagct 3180gccccccgag gagagagccc agaacgtgac caggctgctg agaaagcagg ccgaggaagt 3240gaataccgag tgggagaagc tgaatctgca cagcgccgac tggcagagaa agatcgacga 3300gaccctggag agactccagg aactgcagga agccaccgac gagctggacc tgaagctgag 3360acaggccgaa gtgatcaagg gcagctggca gcctgtgggc gatctgctga tcgactccct 3420gcaggatcac ctggagaaag tgaaggccct gcggggcgag atcgcccccc tgaaggagaa 3480tgtgagccac gtgaacgacc tggccagaca gctgaccacc ctgggcatcc agctgagccc 3540ctacaacctg agcacactgg aggatctgaa cacccggtgg aaactgctgc aggtggccgt 3600ggaggataga gtgaggcagc tgcacgaagc ccacagagac ttcggccctg cctcccagca 3660cttcctgagc accagcgtgc agggcccctg ggagagagcc atctccccca acaaagtgcc 3720ctactacatc aaccacgaga cccagaccac ctgctgggac caccctaaga tgaccgagct 3780gtatcagagc ctggccgacc tgaacaatgt gcggttcagc gcctacagaa ccgccatgaa 3840gctgcggaga ctgcagaagg ccctgtgcct ggatctgctg agcctgagcg ccgcctgcga 3900cgccctggac cagcacaacc tgaagcagaa tgaccagccc atggacatcc tgcagatcat 3960caactgcctg accacaatct acgaccggct ggaacaggag cacaacaacc tggtgaatgt 4020gcccctgtgc gtggacatgt gcctgaattg gctgctgaac gtgtacgaca ccggcaggac 4080cggcagaatc cgcgtgctga gcttcaagac cggcatcatc agcctgtgca aggcccacct 4140ggaggataag taccgctacc tgttcaagca ggtggccagc agcaccggct tctgcgatca 4200gaggagactg ggcctgctgc tgcacgatag catccagatc cctaggcagc tgggcgaagt 4260ggccagcttt ggcggcagca acatcgagcc ctctgtgagg agctgcttcc agttcgccaa 4320caacaagccc gagatcgagg ccgccctgtt cctggactgg atgaggctgg agcctcagag 4380catggtgtgg ctgcctgtgc tgcacagagt ggccgccgcc gagaccgcca agcaccaggc 4440caagtgcaat atctgcaagg agtgccccat catcggcttc cggtacagga gcctgaagca 4500cttcaactac gacatctgcc agagctgctt tttcagcggc agagtggcca agggccacaa 4560aatgcactac cccatggtgg agtactgcac ccccaccacc tccggcgagg atgtgagaga 4620cttcgccaaa gtgctgaaga ataagttccg gaccaagcgg tactttgcca agcaccccag 4680gatgggctac ctgcccgtgc agaccgtgct ggaaggcgac aacatggaga cctgatgagg 4740agctcgagag gcctaataaa gagctcagat gcatcgatca gagtgtgttg gttttttgtg 4800tgagatctag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 4860cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt 4920gagcgagcga gcgcgcagag agggagtggc caa 4953 SEQ IDDNA sequence of AAV-hCK-Hopti-Dys3837 gene expression cassette NO: 10ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctagatc tgaattcggt accccactac gggtttaggc 180tgcccatgta aggaggcaag gcctggggac acccgagatg cctggttata attaacccag 240acatgtggct gccccccccc cccccaacac ctgctgcctc taaaaataac cctgtccctg 300gtggatcccc tgcatgcgaa gatcttcgaa caaggctgtg ggggactgag ggcaggctgt 360aacaggcttg ggggccaggg cttatacgtg cctgggactc ccaaagtatt actgttccat 420gttcccggcg aagggccagc tgtcccccgc cagctagact cagcacttag tttaggaacc 480agtgagcaag tcagcccttg gggcagccca tacaaggcca tggggctggg caagctgcac 540gcctgggtcc ggggtgggca cggtgcccgg gcaacgagct gaaagctcat ctgctctcag 600gggcccctcc ctggggacag cccctcctgg ctagtcacac cctgtaggct cctctatata 660acccaggggc acaggggctg ccctcattct accaccacct ccacagcaca gacagacact 720caggagccag ccagcgtcga gcggccgatc cgccaccatg ctttggtggg aggaagtgga 780ggactgctac gagagagagg acgtgcagaa gaaaaccttc accaagtggg tgaacgccca 840gttcagcaag ttcggcaagc agcacatcga gaacctgttc agcgacctgc aggatggcag 900gagactgctg gacctgctgg agggcctgac cggccagaag ctgcccaagg agaagggcag 960caccagagtg cacgccctga acaacgtgaa caaggccctg agagtgctgc agaacaacaa 1020cgtggacctg gtgaacatcg gcagcaccga catcgtggac ggcaaccaca agctgaccct 1080gggcctgatc tggaacatca tcctgcactg gcaggtgaag aacgtgatga agaacatcat 1140ggccggcctg cagcagacca acagcgagaa gatcctgctg agctgggtga ggcagagcac 1200cagaaactac ccccaggtga acgtgatcaa cttcaccacc tcctggagcg acggcctggc 1260cctgaacgcc ctgatccaca gccacagacc cgacctgttc gactggaaca gcgtggtgtg 1320tcagcagagc gccacccaga gactggagca cgccttcaac atcgccagat accagctggg 1380catcgagaag ctgctggacc ccgaggacgt ggacaccacc taccccgaca agaaaagcat 1440cctcatgtac attaccagcc tgttccaggt gctgccccag caggtgtcca tcgaggccat 1500ccaggaagtg gaaatgctgc ccaggccccc caaagtgacc aaggaggagc acttccagct 1560gcaccaccag atgcactaca gccagcagat cacagtgagc ctggcccagg gctatgagag 1620aaccagcagc cccaagccca gattcaagag ctacgcctac acccaggccg cctacgtgac 1680cacctccgac cccaccagaa gccccttccc cagccagcac ctggaggccc ccgaggacaa 1740gagcttcggc agcagcctga tggagagcga agtgaacctg gacagatacc agaccgccct 1800ggaggaagtg ctgtcctggc tgctgagcgc cgaggacacc ctgcaggccc agggcgagat 1860cagcaacgac gtggaagtgg tgaaggacca gttccacacc cacgagggct acatgatgga 1920tctgaccgcc caccagggca gagtgggcaa tatcctgcag ctgggcagca agctgatcgg 1980caccggcaag ctgagcgagg acgaggagac cgaagtgcag gagcagatga acctgctgaa 2040cagcagatgg gagtgcctga gagtggccag catggagaag cagagcaacc tgcacagagt 2100gctgatggac ctgcagaacc agaagctgaa ggagctgaac gactggctga ccaagaccga 2160ggagcggacc agaaagatgg aggaggagcc cctgggcccc gacctggagg acctgaagag 2220acaggtgcag cagcacaaag tgctgcagga ggacctggag caggagcagg tgcgcgtgaa 2280cagcctgacc cacatggtgg tggtcgtgga cgagagcagc ggcgaccacg ccacagccgc 2340cctggaagag cagctgaaag tgctgggcga cagatgggcc aatatttgta ggtggaccga 2400ggacagatgg gtgctgctgc aggacaccca ccgcctgctc cagcagttcc ccctggacct 2460ggagaagttc ctggcctggc tgaccgaggc cgaaaccacc gccaatgtgc tccaggacgc 2520cactagaaag gagaggctgc tggaggacag caagggcgtg aaagagctga tgaagcagtg 2580gcaggatctg cagggcgaaa tcgaggccca caccgacgtg taccacaacc tggacgagaa 2640cagccagaag attctgagga gcctggaggg cagcgacgac gccgtcctgc tccagaggag 2700gctggacaac atgaacttca agtggagcga gctgcggaag aagagcctga acatccggag 2760ccacctggaa gccagcagcg accagtggaa gagactgcac ctgagcctgc aggagctgct 2820ggtgtggctg cagctgaagg acgacgagct gagcagacag gcccccatcg gcggcgactt 2880ccccgccgtg cagaagcaga acgacgtgca ccgggccttc aagagggagc tgaaaaccaa 2940ggaacccgtg atcatgagca ccctggagac agtgcggatc ttcctgaccg agcagcccct 3000ggagggactg gagaagctgt accaggagcc cagagagctg ccccccgagg agagagccca 3060gaacgtgacc aggctgctga gaaagcaggc cgaggaagtg aataccgagt gggagaagct 3120gaatctgcac agcgccgact ggcagagaaa gatcgacgag accctggaga gactccagga 3180actgcaggaa gccaccgacg agctggacct gaagctgaga caggccgaag tgatcaaggg 3240cagctggcag cctgtgggcg atctgctgat cgactccctg caggatcacc tggagaaagt 3300gaaggccctg cggggcgaga tcgcccccct gaaggagaat gtgagccacg tgaacgacct 3360ggccagacag ctgaccaccc tgggcatcca gctgagcccc tacaacctga gcacactgga 3420ggatctgaac acccggtgga aactgctgca ggtggccgtg gaggatagag tgaggcagct 3480gcacgaagcc cacagagact tcggccctgc ctcccagcac ttcctgagca ccagcgtgca 3540gggcccctgg gagagagcca tctcccccaa caaagtgccc tactacatca accacgagac 3600ccagaccacc tgctgggacc accctaagat gaccgagctg tatcagagcc tggccgacct 3660gaacaatgtg cggttcagcg cctacagaac cgccatgaag ctgcggagac tgcagaaggc 3720cctgtgcctg gatctgctga gcctgagcgc cgcctgcgac gccctggacc agcacaacct 3780gaagcagaat gaccagccca tggacatcct gcagatcatc aactgcctga ccacaatcta 3840cgaccggctg gaacaggagc acaacaacct ggtgaatgtg cccctgtgcg tggacatgtg 3900cctgaattgg ctgctgaacg tgtacgacac cggcaggacc ggcagaatcc gcgtgctgag 3960cttcaagacc ggcatcatca gcctgtgcaa ggcccacctg gaggataagt accgctacct 4020gttcaagcag gtggccagca gcaccggctt ctgcgatcag aggagactgg gcctgctgct 4080gcacgatagc atccagatcc ctaggcagct gggcgaagtg gccagctttg gcggcagcaa 4140catcgagccc tctgtgagga gctgcttcca gttcgccaac aacaagcccg agatcgaggc 4200cgccctgttc ctggactgga tgaggctgga gcctcagagc atggtgtggc tgcctgtgct 4260gcacagagtg gccgccgccg agaccgccaa gcaccaggcc aagtgcaata tctgcaagga 4320gtgccccatc atcggcttcc ggtacaggag cctgaagcac ttcaactacg acatctgcca 4380gagctgcttt ttcagcggca gagtggccaa gggccacaaa atgcactacc ccatggtgga 4440gtactgcacc cccaccacct ccggcgagga tgtgagagac ttcgccaaag tgctgaagaa 4500taagttccgg accaagcggt actttgccaa gcaccccagg atgggctacc tgcccgtgca 4560gaccgtgctg gaaggcgaca acatggagac ctgatgagga gctcgagagg cctaataaag 4620agctcagatg catcgatcag agtgtgttgg ttttttgtgt gagatctagg aacccctagt 4680gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg cccgggcaaa 4740gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga 4800gggagtggcc aa 4812 SEQ IDDNA sequence of AAV-hCKplus-Hopti-Dys3837 gene expression cassetteNO: 11 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc60 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctagatc tgaattcggt accccactac gggtctaggc 180tgcccatgta aggaggcaag gcctggggac acccgagatg cctggttata attaacccca 240acacctgctg cccccccccc cccaacacct gctgcctcta aaaataaccc tgtccctggt 300ggatcccctg catgccccac tacgggttta ggctgcccat gtaaggaggc aaggcctggg 360gacacccgag atgcctggtt ataattaacc cagacatgtg gctgcccccc ccccccccaa 420cacctgctgc ctctaaaaat aaccctgtcc ctggtggatc ccctgcatgc gaagatcttc 480gaacaaggct gtgggggact gagggcaggc tgtaacaggc ttgggggcca gggcttatac 540gtgcctggga ctcccaaagt attactgttc catgttcccg gcgaagggcc agctgtcccc 600cgccagctag actcagcact tagtttagga accagtgagc aagtcagccc ttggggcagc 660ccatacaagg ccatggggct gggcaagctg cacgcctggg tccggggtgg gcacggtgcc 720cgggcaacga gctgaaagct catctgctct caggggcccc tccctgggga cagcccctcc 780tggctagtca caccctgtag gctcctctat ataacccagg ggcacagggg ctgccctcat 840tctaccacca cctccacagc acagacagac actcaggagc cagccagcgt cgagcggccg 900atccgccacc atgctttggt gggaggaagt ggaggactgc tacgagagag aggacgtgca 960gaagaaaacc ttcaccaagt gggtgaacgc ccagttcagc aagttcggca agcagcacat 1020cgagaacctg ttcagcgacc tgcaggatgg caggagactg ctggacctgc tggagggcct 1080gaccggccag aagctgccca aggagaaggg cagcaccaga gtgcacgccc tgaacaacgt 1140gaacaaggcc ctgagagtgc tgcagaacaa caacgtggac ctggtgaaca tcggcagcac 1200cgacatcgtg gacggcaacc acaagctgac cctgggcctg atctggaaca tcatcctgca 1260ctggcaggtg aagaacgtga tgaagaacat catggccggc ctgcagcaga ccaacagcga 1320gaagatcctg ctgagctggg tgaggcagag caccagaaac tacccccagg tgaacgtgat 1380caacttcacc acctcctgga gcgacggcct ggccctgaac gccctgatcc acagccacag 1440acccgacctg ttcgactgga acagcgtggt gtgtcagcag agcgccaccc agagactgga 1500gcacgccttc aacatcgcca gataccagct gggcatcgag aagctgctgg accccgagga 1560cgtggacacc acctaccccg acaagaaaag catcctcatg tacattacca gcctgttcca 1620ggtgctgccc cagcaggtgt ccatcgaggc catccaggaa gtggaaatgc tgcccaggcc 1680ccccaaagtg accaaggagg agcacttcca gctgcaccac cagatgcact acagccagca 1740gatcacagtg agcctggccc agggctatga gagaaccagc agccccaagc ccagattcaa 1800gagctacgcc tacacccagg ccgcctacgt gaccacctcc gaccccacca gaagcccctt 1860ccccagccag cacctggagg cccccgagga caagagcttc ggcagcagcc tgatggagag 1920cgaagtgaac ctggacagat accagaccgc cctggaggaa gtgctgtcct ggctgctgag 1980cgccgaggac accctgcagg cccagggcga gatcagcaac gacgtggaag tggtgaagga 2040ccagttccac acccacgagg gctacatgat ggatctgacc gcccaccagg gcagagtggg 2100caatatcctg cagctgggca gcaagctgat cggcaccggc aagctgagcg aggacgagga 2160gaccgaagtg caggagcaga tgaacctgct gaacagcaga tgggagtgcc tgagagtggc 2220cagcatggag aagcagagca acctgcacag agtgctgatg gacctgcaga accagaagct 2280gaaggagctg aacgactggc tgaccaagac cgaggagcgg accagaaaga tggaggagga 2340gcccctgggc cccgacctgg aggacctgaa gagacaggtg cagcagcaca aagtgctgca 2400ggaggacctg gagcaggagc aggtgcgcgt gaacagcctg acccacatgg tggtggtcgt 2460ggacgagagc agcggcgacc acgccacagc cgccctggaa gagcagctga aagtgctggg 2520cgacagatgg gccaatattt gtaggtggac cgaggacaga tgggtgctgc tgcaggacac 2580ccaccgcctg ctccagcagt tccccctgga cctggagaag ttcctggcct ggctgaccga 2640ggccgaaacc accgccaatg tgctccagga cgccactaga aaggagaggc tgctggagga 2700cagcaagggc gtgaaagagc tgatgaagca gtggcaggat ctgcagggcg aaatcgaggc 2760ccacaccgac gtgtaccaca acctggacga gaacagccag aagattctga ggagcctgga 2820gggcagcgac gacgccgtcc tgctccagag gaggctggac aacatgaact tcaagtggag 2880cgagctgcgg aagaagagcc tgaacatccg gagccacctg gaagccagca gcgaccagtg 2940gaagagactg cacctgagcc tgcaggagct gctggtgtgg ctgcagctga aggacgacga 3000gctgagcaga caggccccca tcggcggcga cttccccgcc gtgcagaagc agaacgacgt 3060gcaccgggcc ttcaagaggg agctgaaaac caaggaaccc gtgatcatga gcaccctgga 3120gacagtgcgg atcttcctga ccgagcagcc cctggaggga ctggagaagc tgtaccagga 3180gcccagagag ctgccccccg aggagagagc ccagaacgtg accaggctgc tgagaaagca 3240ggccgaggaa gtgaataccg agtgggagaa gctgaatctg cacagcgccg actggcagag 3300aaagatcgac gagaccctgg agagactcca ggaactgcag gaagccaccg acgagctgga 3360cctgaagctg agacaggccg aagtgatcaa gggcagctgg cagcctgtgg gcgatctgct 3420gatcgactcc ctgcaggatc acctggagaa agtgaaggcc ctgcggggcg agatcgcccc 3480cctgaaggag aatgtgagcc acgtgaacga cctggccaga cagctgacca ccctgggcat 3540ccagctgagc ccctacaacc tgagcacact ggaggatctg aacacccggt ggaaactgct 3600gcaggtggcc gtggaggata gagtgaggca gctgcacgaa gcccacagag acttcggccc 3660tgcctcccag cacttcctga gcaccagcgt gcagggcccc tgggagagag ccatctcccc 3720caacaaagtg ccctactaca tcaaccacga gacccagacc acctgctggg accaccctaa 3780gatgaccgag ctgtatcaga gcctggccga cctgaacaat gtgcggttca gcgcctacag 3840aaccgccatg aagctgcgga gactgcagaa ggccctgtgc ctggatctgc tgagcctgag 3900cgccgcctgc gacgccctgg accagcacaa cctgaagcag aatgaccagc ccatggacat 3960cctgcagatc atcaactgcc tgaccacaat ctacgaccgg ctggaacagg agcacaacaa 4020cctggtgaat gtgcccctgt gcgtggacat gtgcctgaat tggctgctga acgtgtacga 4080caccggcagg accggcagaa tccgcgtgct gagcttcaag accggcatca tcagcctgtg 4140caaggcccac ctggaggata agtaccgcta cctgttcaag caggtggcca gcagcaccgg 4200cttctgcgat cagaggagac tgggcctgct gctgcacgat agcatccaga tccctaggca 4260gctgggcgaa gtggccagct ttggcggcag caacatcgag ccctctgtga ggagctgctt 4320ccagttcgcc aacaacaagc ccgagatcga ggccgccctg ttcctggact ggatgaggct 4380ggagcctcag agcatggtgt ggctgcctgt gctgcacaga gtggccgccg ccgagaccgc 4440caagcaccag gccaagtgca atatctgcaa ggagtgcccc atcatcggct tccggtacag 4500gagcctgaag cacttcaact acgacatctg ccagagctgc tttttcagcg gcagagtggc 4560caagggccac aaaatgcact accccatggt ggagtactgc acccccacca cctccggcga 4620ggatgtgaga gacttcgcca aagtgctgaa gaataagttc cggaccaagc ggtactttgc 4680caagcacccc aggatgggct acctgcccgt gcagaccgtg ctggaaggcg acaacatgga 4740gacctgatga ggagctcgag aggcctaata aagagctcag atgcatcgat cagagtgtgt 4800tggttttttg tgtgagatct aggaacccct agtgatggag ttggccactc cctctctgcg 4860cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg 4920cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaa 4965 SEQ IDDNA sequence of AAV-hCK-Copti-Dys3978 gene expression cassette NO: 12ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctcagat ctgaattcgg taccccacta cgggtttagg 180ctgcccatgt aaggaggcaa ggcctgggga cacccgagat gcctggttat aattaaccca 240gacatgtggc tgcccccccc ccccccaaca cctgctgcct ctaaaaataa ccctgtccct 300ggtggatccc ctgcatgcga agatcttcga acaaggctgt gggggactga gggcaggctg 360taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat tactgttcca 420tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta gtttaggaac 480cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg gcaagctgca 540cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaaagctca tctgctctca 600ggggcccctc cctggggaca gcccctcctg gctagtcaca ccctgtaggc tcctctatat 660aacccagggg cacaggggct gccctcattc taccaccacc tccacagcac agacagacac 720tcaggagcca gccagcgtcg agcggccgcc accatgcttt ggtgggagga agtggaggac 780tgctacgagc gggaggacgt gcagaagaaa accttcacca agtgggtgaa cgcccagttc 840agcaagttcg gcaagcagca catcgagaac ctgttcagcg acctgcagga cggcaggcgg 900ctgctggacc tcctggaagg cctgaccggc cagaagctgc ccaaagagaa gggcagcacc 960agggtgcacg ccctgaacaa cgtgaacaag gccctgaggg tgctgcagaa caacaacgtg 1020gacctggtga acatcggcag caccgacatc gtggacggca accacaagct gaccctgggc 1080ctgatctgga acatcatcct gcactggcag gtcaagaacg tgatgaagaa catcatggcc 1140ggcctgcagc agaccaacag cgagaagatc ctgctgtcct gggtgcggca gagcaccagg 1200aactaccccc aggtcaacgt gatcaacttc accacctctt ggagcgacgg cctggccctg 1260aacgccctga tccacagcca caggcccgac ctgttcgact ggaacagcgt ggtgtgccag 1320cagagcgcca cccagaggct ggaacacgcc ttcaacatcg ccagatacca gctgggcatc 1380gagaagctgc tggatcccga ggacgtggac accacctacc ccgacaagaa aagcatcctc 1440atgtacatca ccagcctgtt ccaggtgctg ccccagcagg tgtccatcga ggccatccag 1500gaagtggaga tgctgcccag gccccccaag gtcaccaaag aggaacactt ccagctgcac 1560caccagatgc actacagcca gcagatcacc gtgagcctgg cccagggcta cgagaggacc 1620agcagcccca agcccaggtt caagagctac gcctacaccc aggccgccta cgtgaccacc 1680tccgacccca ccaggtcccc cttccccagc cagcatctcg aagcccccga ggacaagagc 1740ttcggcagca gcctgatgga aagcgaggtg aacctggaca gataccagac cgccctggaa 1800gaagtgctgt cttggctgct gtccgccgag gacaccctgc aggcccaggg cgagatcagc 1860aacgacgtgg aggtcgtgaa ggaccagttc cacacccacg agggctacat gatggacctg 1920accgcccacc agggcagagt gggcaacatc ctgcagctgg gcagcaagct gatcggcacc 1980ggcaagctgt ccgaggacga ggaaaccgag gtgcaggaac agatgaacct gctgaacagc 2040agatgggagt gcctgagggt ggccagcatg gaaaagcaga gcaacctgca cagggtgctg 2100atggatctgc agaaccagaa gctcaaagag ctgaacgact ggctgaccaa gaccgaggaa 2160aggacccgga agatggaaga ggaacccctg ggccccgatc togaagatct gaagaggcag 2220gtgcagcagc acaaggtgct gcaggaagat ctcgaacagg aacaggtccg ggtcaacagc 2280ctgacccaca tggtcgtggt ggtggacgag agcagcggcg accacgccac cgctgccctg 2340gaagagcagc tgaaggtgct gggcgacaga tgggccaaca tctgccggtg gaccgaggac 2400agatgggtcc tcctgcagga ccagcccgac ctggcccctg gcctgacaac catcggcgcc 2460agccccaccc agaccgtgac cctggtgacc cagcccgtgg tgaccaaaga gaccgccatc 2520agcaagctgg aaatgcccag ctccctgatg ctggaagtgc ccacccacag gctcctccag 2580cagttccccc tggacctgga aaagttcctg gcctggctga ccgaggccga gaccaccgcc 2640aacgtgctgc aggacgccac caggaaagag aggctgctgg aagatagcaa gggcgtgaaa 2700gagctgatga agcagtggca ggacctgcag ggggagattg aggcccacac cgacgtgtac 2760cacaacctgg acgagaacag ccagaaaatc ctgagaagcc tggaaggcag cgacgacgcc 2820gtgctgctgc agaggcggct ggacaacatg aacttcaagt ggagcgagct gaggaagaag 2880agcctgaaca tcaggtccca tctggaagcc agcagcgacc agtggaagag gctgcacctg 2940agcctgcagg aactgctcgt ctggctgcag ctgaaagacg acgagctgtc caggcaggcc 3000cccatcggcg gcgacttccc cgccgtgcag aaacagaacg acgtgcacag ggccttcaag 3060cgggagctga aaaccaaaga gcccgtgatc atgagcaccc tggaaaccgt gaggatcttc 3120ctgaccgagc agcccctgga aggactggaa aagctgtacc aggaacccag agagctgccc 3180cccgaggaac gggcccagaa cgtgaccagg ctgctgagaa agcaggccga ggaagtgaac 3240accgagtggg agaagctgaa cctgcactcc gccgactggc agaggaagat cgacgagacc 3300ctggaaaggc tccaggaact gcaggaagcc accgacgagc tggacctgaa gctgagacag 3360gccgaggtga tcaagggcag ctggcagccc gtgggcgacc tgctgatcga ctccctgcag 3420gatcacctgg aaaaagtgaa ggccctgcgg ggcgagatcg cccccctgaa agagaacgtc 3480agccacgtca acgacctggc caggcagctg accaccctgg gcatccagct gtccccctac 3540aacctgtcca ccctggaaga tctgaacaca aggtggaagc tgctgcaggt ggccgtggag 3600gacagagtga ggcagctgca cgaggcccac agggacttcg gccctgcctc ccagcacttc 3660ctgagcacca gcgtgcaggg cccctgggag agggccatct cccccaacaa ggtgccctac 3720tacatcaacc acgagaccca gaccacctgc tgggaccacc ctaagatgac cgagctgtac 3780cagtccctgg ccgacctgaa caatgtgcgg ttcagcgcct accggaccgc catgaagctg 3840aggcggctgc agaaagccct gtgcctggat ctgctgtccc tgagcgccgc ctgcgacgcc 3900ctggaccagc acaacctgaa gcagaacgac cagcccatgg atatcctgca gatcatcaac 3960tgtctgacca ccatctacga caggctggaa caggaacaca acaacctggt caacgtgccc 4020ctgtgcgtgg acatgtgcct gaactggctg ctgaacgtgt acgacaccgg caggaccggc 4080cggatcaggg tgctgtcctt caagaccggc atcatcagcc tgtgcaaggc ccacctggaa 4140gataagtacc gctacctgtt caagcaggtg gccagctcta ccggcttctg cgaccagcgg 4200aggctgggcc tgctgctgca cgacagcatc cagatccccc ggcagctggg cgaggtggcc 4260tccttcggcg gcagcaacat cgagcccagc gtgcggagct gcttccagtt cgccaacaac 4320aagcccgaga tcgaggccgc cctgttcctg gactggatgc ggctggaacc ccagagcatg 4380gtctggctgc ccgtgctgca cagagtggct gccgccgaga ccgccaagca ccaggccaag 4440tgcaacatct gcaaagagtg ccccatcatc ggcttcaggt acagaagcct gaagcacttc 4500aactacgaca tctgccagag ctgtttcttc agcggcaggg tggccaaggg ccacaaaatg 4560cactacccca tggtggagta ctgcaccccc accacctccg gcgaggacgt gagggacttc 4620gccaaggtgc tgaagaataa gttccggacc aagcggtact tcgccaaaca ccccaggatg 4680ggctacctgc ccgtgcagac cgtgctggaa ggcgacaaca tggaaacctg ataacacgcg 4740tcgactcgag aggcctaata aagagctcag atgcatcgat cagagtgtgt tggttttttg 4800tgtgagatct aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4860ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 4920gtgagcgagc gagcgcgcag agagggagtg gccaa  4955 SEQ IDAmino acid sequence of AAV9 capsid VP1 protein NO: 13MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD 60KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 120AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 180SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI 240TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 300LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH 360EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV 420PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP 480GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS 540LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG 600ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT 660AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV 720YSEPRPIGTR YLTRNL 736 SEQ ID Left AAV2 ITR NO: 14ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcct 145 SEQ ID Right AAV2 ITR NO: 15aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 120gagcgcgcag agagggagtg gccaa 145 SEQ IDDNA sequence of synthetic muscle-specific enhancer and promoter NO: 16ccactacggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 60ggttataatt aacccagaca tgtggctgcc cccccccccc ccaacacctg ctgcctctaa 120aaataaccct gtccctggtg gatcccctgc atgcgaagat cttcgaacaa ggctgtgggg 180gactgagggc aggctgtaac aggcttgggg gccagggctt atacgtgcct gggactccca 240aagtattact gttccatgtt cccggcgaag ggccagctgt cccccgccag ctagactcag 300cacttagttt aggaaccagt gagcaagtca gcccttgggg cagcccatac aaggccatgg 360ggctgggcaa gctgcacgcc tgggtccggg gtgggcacgg tgcccgggca acgagctgaa 420agctcatctg ctctcagggg cccctccctg gggacagccc ctcctggcta gtcacaccct 480gtaggctcct ctatataacc caggggcaca ggggctgccc tcattctacc accacctcca 540cagcacagac agacactcag gagccagcca gcgtcga 577 SEQ IDDNA sequence of transcription terminator NO: 17aggcctaata aagagctcag atgcatcgat cagagtgtgt tggttttttg tgtg 54 SEQ IDDNA sequence of AAV9.hCK.Hopti-Dys3978.spA vector genome NO: 18ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctcagat ctgaattcgg taccccacta cgggtctagg 180ctgcccatgt aaggaggcaa ggcctgggga cacccgagat gcctggttat aattaaccca 240gacatgtggc tgcccccccc ccccccaaca cctgctgcct ctaaaaataa ccctgtccct 300ggtggatccc ctgcatgcga agatcttcga acaaggctgt gggggactga gggcaggctg 360taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat tactgttcca 420tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta gtttaggaac 480cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg gcaagctgca 540cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaaagctca tctgctctca 600ggggcccctc cctggggaca gcccctcctg gctagtcaca ccctgtaggc tcctctatat 660aacccagggg cacaggggct gccctcattc taccaccacc tccacagcac agacagacac 720tcaggagcca gccagcgtcg agcggccgat ccgccaccat gctttggtgg gaggaagtgg 780aggactgcta cgagagagag gacgtgcaga agaaaacctt caccaagtgg gtgaacgccc 840agttcagcaa gttcggcaag cagcacatcg agaacctgtt cagcgacctg caggatggca 900ggagactgct ggacctgctg gagggcctga ccggccagaa gctgcccaag gagaagggca 960gcaccagagt gcacgccctg aacaacgtga acaaggccct gagagtgctg cagaacaaca 1020acgtggacct ggtgaacatc ggcagcaccg acatcgtgga cggcaaccac aagctgaccc 1080tgggcctgat ctggaacatc atcctgcact ggcaggtgaa gaacgtgatg aagaacatca 1140tggccggcct gcagcagacc aacagcgaga agatcctgct gagctgggtg aggcagagca 1200ccagaaacta cccccaggtg aacgtgatca acttcaccac ctcctggagc gacggcctgg 1260ccctgaacgc cctgatccac agccacagac ccgacctgtt cgactggaac agcgtggtgt 1320gtcagcagag cgccacccag agactggagc acgccttcaa catcgccaga taccagctgg 1380gcatcgagaa gctgctggac cccgaggacg tggacaccac ctaccccgac aagaaaagca 1440tcctcatgta cattaccagc ctgttccagg tgctgcccca gcaggtgtcc atcgaggcca 1500tccaggaagt ggaaatgctg cccaggcccc ccaaagtgac caaggaggag cacttccagc 1560tgcaccacca gatgcactac agccagcaga tcacagtgag cctggcccag ggctatgaga 1620gaaccagcag ccccaagccc agattcaaga gctacgccta cacccaggcc gcctacgtga 1680ccacctccga ccccaccaga agccccttcc ccagccagca cctggaggcc cccgaggaca 1740agagcttcgg cagcagcctg atggagagcg aagtgaacct ggacagatac cagaccgccc 1800tggaggaagt gctgtcctgg ctgctgagcg ccgaggacac cctgcaggcc cagggcgaga 1860tcagcaacga cgtggaagtg gtgaaggacc agttccacac ccacgagggc tacatgatgg 1920atctgaccgc ccaccagggc agagtgggca atatcctgca gctgggcagc aagctgatcg 1980gcaccggcaa gctgagcgag gacgaggaga ccgaagtgca ggagcagatg aacctgctga 2040acagcagatg ggagtgcctg agagtggcca gcatggagaa gcagagcaac ctgcacagag 2100tgctgatgga cctgcagaac cagaagctga aggagctgaa cgactggctg accaagaccg 2160aggagcggac cagaaagatg gaggaggagc ccctgggccc cgacctggag gacctgaaga 2220gacaggtgca gcagcacaaa gtgctgcagg aggacctgga gcaggagcag gtgcgcgtga 2280acagcctgac ccacatggtg gtggtcgtgg acgagagcag cggcgaccac gccacagccg 2340ccctggaaga gcagctgaaa gtgctgggcg acagatgggc caatatttgt aggtggaccg 2400aggacagatg ggtgctgctg caggaccagc ccgacctggc ccctggcctg accaccatcg 2460gcgccagccc cacccagacc gtgaccctgg tgacccagcc cgtggtgaca aaggagaccg 2520ccatcagcaa gctggagatg cccagctccc tgatgctgga agtgcccacc caccgcctgc 2580tccagcagtt ccccctggac ctggagaagt tcctggcctg gctgaccgag gccgaaacca 2640ccgccaatgt gctccaggac gccactagaa aggagaggct gctggaggac agcaagggcg 2700tgaaagagct gatgaagcag tggcaggatc tgcagggcga aatcgaggcc cacaccgacg 2760tgtaccacaa cctggacgag aacagccaga agattctgag gagcctggag ggcagcgacg 2820acgccgtcct gctccagagg aggctggaca acatgaactt caagtggagc gagctgcgga 2880agaagagcct gaacatccgg agccacctgg aagccagcag cgaccagtgg aagagactgc 2940acctgagcct gcaggagctg ctggtgtggc tgcagctgaa ggacgacgag ctgagcagac 3000aggcccccat cggcggcgac ttccccgccg tgcagaagca gaacgacgtg caccgggcct 3060tcaagaggga gctgaaaacc aaggaacccg tgatcatgag caccctggag acagtgcgga 3120tcttcctgac cgagcagccc ctggagggac tggagaagct gtaccaggag cccagagagc 3180tgccccccga ggagagagcc cagaacgtga ccaggctgct gagaaagcag gccgaggaag 3240tgaataccga gtgggagaag ctgaatctgc acagcgccga ctggcagaga aagatcgacg 3300agaccctgga gagactccag gaactgcagg aagccaccga cgagctggac ctgaagctga 3360gacaggccga agtgatcaag ggcagctggc agcctgtggg cgatctgctg atcgactccc 3420tgcaggatca cctggagaaa gtgaaggccc tgcggggcga gatcgccccc ctgaaggaga 3480atgtgagcca cgtgaacgac ctggccagac agctgaccac cctgggcatc cagctgagcc 3540cctacaacct gagcacactg gaggatctga acacccggtg gaaactgctg caggtggccg 3600tggaggatag agtgaggcag ctgcacgaag cccacagaga cttcggccct gcctcccagc 3660acttcctgag caccagcgtg cagggcccct gggagagagc catctccccc aacaaagtgc 3720cctactacat caaccacgag acccagacca cctgctggga ccaccctaag atgaccgagc 3780tgtatcagag cctggccgac ctgaacaatg tgcggttcag cgcctacaga accgccatga 3840agctgcggag actgcagaag gccctgtgcc tggatctgct gagcctgagc gccgcctgcg 3900acgccctgga ccagcacaac ctgaagcaga atgaccagcc catggacatc ctgcagatca 3960tcaactgcct gaccacaatc tacgaccggc tggaacagga gcacaacaac ctggtgaatg 4020tgcccctgtg cgtggacatg tgcctgaatt ggctgctgaa cgtgtacgac accggcagga 4080ccggcagaat ccgcgtgctg agcttcaaga ccggcatcat cagcctgtgc aaggcccacc 4140tggaggataa gtaccgctac ctgttcaagc aggtggccag cagcaccggc ttctgcgatc 4200agaggagact gggcctgctg ctgcacgata gcatccagat ccctaggcag ctgggcgaag 4260tggccagctt tggcggcagc aacatcgagc cctctgtgag gagctgcttc cagttcgcca 4320acaacaagcc cgagatcgag gccgccctgt tcctggactg gatgaggctg gagcctcaga 4380gcatggtgtg gctgcctgtg ctgcacagag tggccgccgc cgagaccgcc aagcaccagg 4440ccaagtgcaa tatctgcaag gagtgcccca tcatcggctt ccggtacagg agcctgaagc 4500acttcaacta cgacatctgc cagagctgct ttttcagcgg cagagtggcc aagggccaca 4560aaatgcacta ccccatggtg gagtactgca cccccaccac ctccggcgag gatgtgagag 4620acttcgccaa agtgctgaag aataagttcc ggaccaagcg gtactttgcc aagcacccca 4680ggatgggcta cctgcccgtg cagaccgtgc tggaaggcga caacatggag acctgatgag 4740gagctcgaga ggcctaataa agagctcaga tgcatcgatc agagtgtgtt ggttttttgt 4800gtgagatctg aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4860ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 4920gtgagcgagc gagcgcgcag agagggagtg gccaa  4955 SEQ IDDNA sequence of PCR forward primer for mini-dystrophin gene NO: 19ccaacaaagt gccctactac atc 23 SEQ IDDNA sequence of PCR reverse primer for mini-dystrophin gene NO: 20ggttgtgctg gtccagggcg t 21 SEQ IDDNA sequence of probe for mini-dystrophin gene NO: 21ccgagctgta tcagagcctg gcc 23 SEQ IDDNA sequence of PCR forward primer for rat HPRT1 gene NO: 22gcgaaagtgg aaaagccaag t 21 SEQ IDDNA sequence of PCR reverse primer for rat HPRT1 gene NO: 23gccacatcaa caggactctt gtag 24 SEQ IDDNA sequence of probe for rat HPRT1 gene NO: 24caaagcctaa aagacagcgg caagttgaat 30 SEQ IDAmino acid sequence of human muscle dystrophin (Dp427m isoform) NO: 25MLWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ 60KLPKEKGSTR VHALNNVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV 120KNVMKNIMAG LQQTNSEKIL LSWVRQSTRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL 180FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP 240QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA 300YTQAAYVTTS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAED 360TLQAQGEISN DVEVVKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV 420QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG 480PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMVVVVDES SGDHATAALE EQLKVLGDRW 540ANICRWTEDR WVLLQDILLK WQRLTEEQCL FSAWLSEKED AVNKIHTTGF KDQNEMLSSL 600QKLAVLKADL EKKKQSMGKL YSLKQDLLST LKNKSVTQKT EAWLDNFARC WDNLVQKLEK 660STAQISQAVT TTQPSLTQTT VMETVTTVTT REQILVKHAQ EELPPPPPQK KRQITVDSEI 720RKRLDVDITE LHSWITRSEA VLQSPEFAIF RKEGNFSDLK EKVNAIEREK AEKFRKLQDA 780SRSAQALVEQ MVNEGVNADS IKQASEQLNS RWIEFCQLLS ERLNWLEYQN NIIAFYNQLQ 840QLEQMTTTAE NWLKIQPTTP SEPTAIKSQL KICKDEVNRL SGLQPQIERL KIQSIALKEK 900GQGPMFLDAD FVAFTNHFKQ VESDVQAREK ELQTIFDTLP PMRYQETMSA IRTWVQQSET 960KLSIPQLSVT DYEIMEQRLG ELQALQSSLQ EQQSGLYYLS TTVKEMSKKA PSEISRKYQS 1020EFEEIEGRWK KLSSQLVEHC QKLEEQMNKL RKIQNHIQTL KKWMAEVDVF LKEEWPALGD 1080SEILKKQLKQ CRLLVSDIQT IQPSLNSVNE GGQKIKNEAE PEFASRLETE LKELNTQWDH 1140MCQQVYARKE ALKGGLEKTV SLQKDLSEMH EWMTQAEEEY LERDFEYKTP DELQKAVEEM 1200KRAKEEAQQK EAKVKLLTES VNSVIAQAPP VAQEALKKEL ETLTTNYQWL CTRLNGKCKT 1260LEEVWACWHE LLSYLEKANK WLNEVEFKLK TTENIPGGAE EISEVLDSLE NLMRHSEDNP 1320NQIRILAQTL TDGGVMDELI NEELETFNSR WRELHEEAVR RQKLLEQSIQ SAQETEKSLH 1380LIQESLTFID KQLAAYIADK VDAAQMPQEA QKIQSDLTSH EISLEEMKKH NQGKEAAQRV 1440LSQIDVAQKK LQDVSMKFRL FQKPANFEQR LQESKMILDE VKMHLPALET KSVEQEVVQS 1500QLNHCVNLYK SLSEVKSEVE MVIKTGRQIV QKKQTENPKE LDERVTALKL HYNELGAKVT 1560ERKQQLEKCL KLSRKMRKEM NVLTEWLAAT DMELTKRSAV EGMPSNLDSE VAWGKATQKE 1620IEKQKVHLKS ITEVGEALKT VLGKKETLVE DKLSLLNSNW IAVTSRAEEW LNLLLEYQKH 1680METFDQNVDH ITKWIIQADT LLDESEKKKP QQKEDVLKRL KAELNDIRPK VDSTRDQAAN 1740LMANRGDHCR KLVEPQISEL NHRFAAISHR IKTGKASIPL KELEQFNSDI QKLLEPLEAE 1800IQQGVNLKEE DFNKDMNEDN EGTVKELLQR GDNLQQRITD ERKREEIKIK QQLLQTKHNA 1860LKDLRSQRRK KALEISHQWY QYKRQADDLL KCLDDIEKKL ASLPEPRDER KIKEIDRELQ 1920KKKEELNAVR RQAEGLSEDG AAMAVEPTQI QLSKRWREIE SKFAQFRRLN FAQIHTVREE 1980TMMVMTEDMP LEISYVPSTY LTEITHVSQA LLEVEQLLNA PDLCAKDFED LFKQEESLKN 2040IKDSLQQSSG RIDIIHSKKT AALQSATPVE RVKLQEALSQ LDFQWEKVNK MYKDRQGRFD 2100RSVEKWRRFH YDIKIFNQWL TEAEQFLRKT QIPENWEHAK YKWYLKELQD GIGQRQTVVR 2160TLNATGEEII QQSSKTDASI LQEKLGSLNL RWQEVCKQLS DRKKRLEEQK NILSEFQRDL 2220NEFVLWLEEA DNIASIPLEP GKEQQLKEKL EQVKLLVEEL PLRQGILKQL NETGGPVLVS 2280APISPEEQDK LENKLKQTNL QWIKVSRALP EKQGEIEAQI KDLGQLEKKL EDLEEQLNHL 2340LLWLSPIRNQ LEIYNQPNQE GPFDVQETEI AVQAKQPDVE EILSKGQHLY KEKPATQPVK 2400RKLEDLSSEW KAVNRLLQEL RAKQPDLAPG LTTIGASPTQ TVTLVTQPVV TKETAISKLE 2460MPSSLMLEVP ALADFNRAWT ELTDWLSLLD QVIKSQRVMV GDLEDINEMI IKQKATMQDL 2520EQRRPQLEEL ITAAQNLKNK TSNQEARTII TDRIERIQNQ WDEVQEHLQN RRQQLNEMLK 2580DSTQWLEAKE EAEQVLGQAR AKLESWKEGP YTVDAIQKKI TETKQLAKDL RQWQTNVDVA 2640NDLALKLLRD YSADDTRKVH MITENINASW RSIHKRVSER EAALEETHRL LQQFPLDLEK 2700FLAWLTEAET TANVLQDATR KERLLEDSKG VKELMKQWQD LQGEIEAHTD VYHNLDENSQ 2760KILRSLEGSD DAVLLQRRLD NMNFKWSELR KKSLNIRSHL EASSDQWKRL HLSLQELLVW 2820LQLKDDELSR QAPIGGDFPA VQKQNDVHRA FKRELKTKEP VIMSTLETVR IFLTEQPLEG 2880LEKLYQEPRE LPPEERAQNV TRLLRKQAEE VNTEWEKLNL HSADWQRKID ETLERLQELQ 2940EATDELDLKL RQAEVIKGSW QPVGDLLIDS LQDHLEKVKA LRGEIAPLKE NVSHVNDLAR 3000QLTTLGIQLS PYNLSTLEDL NTRWKLLQVA VEDRVRQLHE AHRDFGPASQ HFLSTSVQGP 3060WERAISPNKV PYYINHETQT TCWDHPKMTE LYQSLADLNN VRFSAYRTAM KLRRLQKALC 3120LDLLSLSAAC DALDQHNLKQ NDQPMDILQI INCLTTIYDR LEQEHNNLVN VPLCVDMCLN 3180WLLNVYDTGR TGRIRVLSFK TGIISLCKAH LEDKYRYLFK QVASSTGFCD QRRLGLLLHD 3240SIQIPRQLGE VASFGGSNIE PSVRSCFQFA NNKPEIEAAL FLDWMRLEPQ SMVWLPVLHR 3300VAAAETAKHQ AKCNICKECP IIGFRYRSLK HFNYDICQSC FFSGRVAKGH KMHYPMVEYC 3360TPTTSGEDVR DFAKVLKNKF RTKRYFAKHP RMGYLPVQTV LEGDNMETPV TLINFWPVDS 3420APASSPQLSH DDTHSRIEHY ASRLAEMENS NGSYLNDSIS PNESIDDEHL LIQHYCQSLN 3480QDSPLSQPRS PAQILISLES EERGELERIL ADLEEENRNL QAEYDRLKQQ HEHKGLSPLP 3540SPPEMMPTSP QSPRDAELIA EAKLLRQHKG RLEARMQILE DHNKQLESQL HRLRQLLEQP 3600QAEAKVNGTT VSSPSTSLQR SDSSQPMLLR VVGSQTSDSM GEEDLLSPPQ DTSTGLEEVM 3660EQLNNSFPSS RGRNTPGKPM REDTM 3685 SEQ IDDNA sequence of non-codon-optimized gene encoding human mini-dystrophin Dys3987NO: 26 atgctttggt gggaagaagt agaggactgt tatgaaagag aagatgttca aaagaaaaca60 ttcacaaaat gggtaaatgc acaattttct aagtttggga agcagcatat tgagaacctc 120ttcagtgacc tacaggatgg gaggcgcctc ctagacctcc togaaggcct gacagggcaa 180aaactgccaa aagaaaaagg atccacaaga gttcatgccc tgaacaatgt caacaaggca 240ctgcgggttt tgcagaacaa taatgttgat ttagtgaata ttggaagtac tgacatcgta 300gatggaaatc ataaactgac tcttggtttg atttggaata taatcctcca ctggcaggtc 360aaaaatgtaa tgaaaaatat catggctgga ttgcaacaaa ccaacagtga aaagattctc 420ctgagctggg tccgacaatc aactcgtaat tatccacagg ttaatgtaat caacttcacc 480accagctggt ctgatggcct ggctttgaat gctctcatcc atagtcatag gccagaccta 540tttgactgga atagtgtggt ttgccagcag tcagccacac aacgactgga acatgcattc 600aacatcgcca gatatcaatt aggcatagag aaactactcg atcctgaaga tgttgatacc 660acctatccag ataagaagtc catcttaatg tacatcacat cactcttcca agttttgcct 720caacaagtga gcattgaagc catccaggaa gtggaaatgt tgccaaggcc acctaaagtg 780actaaagaag aacattttca gttacatcat caaatgcact attctcaaca gatcacggtc 840agtctagcac agggatatga gagaacttct tcccctaagc ctcgattcaa gagctatgcc 900tacacacagg ctgcttatgt caccacctct gaccctacac ggagcccatt tccttcacag 960catttggaag ctcctgaaga caagtcattt ggcagttcat tgatggagag tgaagtaaac 1020ctggaccgtt atcaaacagc tttagaagaa gtattatcgt ggcttctttc tgctgaggac 1080acattgcaag cacaaggaga gatttctaat gatgtggaag tggtgaaaga ccagtttcat 1140actcatgagg ggtacatgat ggatttgaca gcccatcagg gccgggttgg taatattcta 1200caattgggaa gtaagctgat tggaacagga aaattatcag aagatgaaga aactgaagta 1260caagagcaga tgaatctcct aaattcaaga tgggaatgcc tcagggtagc tagcatggaa 1320aaacaaagca atttacatag agttttaatg gatctccaga atcagaaact gaaagagttg 1380aatgactggc taacaaaaac agaagaaaga acaaggaaaa tggaggaaga gcctcttgga 1440cctgatcttg aagacctaaa acgccaagta caacaacata aggtgcttca agaagatcta 1500gaacaagaac aagtcagggt caattctctc actcacatgg tggtggtagt tgatgaatct 1560agtggagatc acgcaactgc tgctttggaa gaacaactta aggtattggg agatcgatgg 1620gcaaacatct gtagatggac agaagaccgc tgggttcttt tacaagacca gcctgaccta 1680gctcctggac tgaccactat tggagcctct cctactcaga ctgttactct ggtgacacaa 1740cctgtggtta ctaaggaaac tgccatctcc aaactagaaa tgccatcttc cttgatgttg 1800gaggtaccta ctcatagatt actgcaacag ttccccctgg acctggaaaa gtttcttgcc 1860tggcttacag aagctgaaac aactgccaat gtcctacagg atgctacccg taaggaaagg 1920ctcctagaag actccaaggg agtaaaagag ctgatgaaac aatggcaaga cctccaaggt 1980gaaattgaag ctcacacaga tgtttatcac aacctggatg aaaacagcca aaaaatcctg 2040agatccctgg aaggttccga tgatgcagtc ctgttacaaa gacgtttgga taacatgaac 2100ttcaagtgga gtgaacttcg gaaaaagtct ctcaacatta ggtcccattt ggaagccagt 2160tctgaccagt ggaagcgtct gcacctttct ctgcaggaac ttctggtgtg gctacagctg 2220aaagatgatg aattaagccg gcaggcacct attggaggcg actttccagc agttcagaag 2280cagaacgatg tacatagggc cttcaagagg gaattgaaaa ctaaagaacc tgtaatcatg 2340agtactcttg agactgtacg aatatttctg acagagcagc ctttggaagg actagagaaa 2400ctctaccagg agcccagaga gctgcctcct gaggagagag cccagaatgt cactcggctt 2460ctacgaaagc aggctgagga ggtcaatact gagtgggaaa aattgaacct gcactccgct 2520gactggcaga gaaaaataga tgagaccctt gaaagactcc aggaacttca agaggccacg 2580gatgagctgg acctcaagct gcgccaagct gaggtgatca agggatcctg gcagcccgtg 2640ggcgatctcc tcattgactc tctccaagat cacctcgaga aagtcaaggc acttcgagga 2700gaaattgcgc ctctgaaaga gaacgtgagc cacgtcaatg accttgctcg ccagcttacc 2760actttgggca ttcagctctc accgtataac ctcagcactc tggaagacct gaacaccaga 2820tggaagcttc tgcaggtggc cgtcgaggac cgagtcaggc agctgcatga agcccacagg 2880gactttggtc cagcatctca gcactttctt tccacgtctg tccagggtcc ctgggagaga 2940gccatctcgc caaacaaagt gccctactat atcaaccacg agactcaaac aacttgctgg 3000gaccatccca aaatgacaga gctctaccag tctttagctg acctgaataa tgtcagattc 3060tcagcttata ggactgccat gaaactccga agactgcaga aggccctttg cttggatctc 3120ttgagcctgt cagctgcatg tgatgccttg gaccagcaca acctcaagca aaatgaccag 3180cccatggata tcctgcagat tattaattgt ttgaccacta tttatgaccg cctggagcaa 3240gagcacaaca atttggtcaa cgtccctctc tgcgtggata tgtgtctgaa ctggctgctg 3300aatgtttatg atacgggacg aacagggagg atccgtgtcc tgtcttttaa aactggcatc 3360atttccctgt gtaaagcaca tttggaagac aagtacagat accttttcaa gcaagtggca 3420agttcaacag gattttgtga ccagcgcagg ctgggcctcc ttctgcatga ttctatccaa 3480attccaagac agttgggtga agttgcatcc tttgggggca gtaacattga gccaagtgtc 3540cggagctgct tccaatttgc taataataag ccagagatcg aagcggccct cttcctagac 3600tggatgagac tggaacccca gtccatggtg tggctgcccg tcctgcacag agtggctgct 3660gcagaaactg ccaagcatca ggccaaatgt aacatctgca aagagtgtcc aatcattgga 3720ttcaggtaca ggagtctaaa gcactttaat tatgacatct gccaaagctg ctttttttct 3780ggtcgagttg caaaaggcca taaaatgcac tatcccatgg tggaatattg cactccgact 3840acatcaggag aagatgttcg agactttgcc aaggtactaa aaaacaaatt tcgaaccaaa 3900aggtattttg cgaagcatcc ccgaatgggc tacctgccag tgcagactgt cttagagggg 3960gacaacatgg aaacttag 3978 SEQ IDAmino acid sequence of human mini-dystrophin protein 43990 NO: 27MVWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ 60KLPKEKGSTR VHALNNVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV 120KNVMKNIMAG LQQTNSEKIL LSWVRQSTRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL 180FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP 240QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA 300YTQAAYVTTS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAED 360TLQAQGEISN DVEVVKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV 420QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG 480PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMVVVVDES SGDHATAALE EQLKVLGDRW 540ANICRWTEDR WVLLQDQPDL APGLTTIGAS PTQTVTLVTQ PVVTKETAIS KLEMPSSLML 600EVPTHRLLQQ FPLDLEKFLA WLTEAETTAN VLQDATRKER LLEDSKGVKE LMKQWQDLQG 660EIEAHTDVYH NLDENSQKIL RSLEGSDDAV LLQRRLDNMN FKWSELRKKS LNIRSHLEAS 720SDQWKRLHLS LQELLVWLQL KDDELSRQAP IGGDFPAVQK QNDVHRAFKR ELKTKEPVIM 780STLETVRIFL TEQPLEGLEK LYQEPRELPP EERAQNVTRL LRKQAEEVNT EWEKLNLHSA 840DWQRKIDETL ERLQELQEAT DELDLKLRQA EVIKGSWQPV GDLLIDSLQD HLEKVKALRG 900EIAPLKENVS HVNDLARQLT TLGIQLSPYN LSTLEDLNTR WKLLQVAVED RVRQLHEAHR 960DFGPASQHFL STSVQGPWER AISPNKVPYY INHETQTTCW DHPKMTELYQ SLADLNNVRF 1020SAYRTAMKLR RLQKALCLDL LSLSAACDAL DQHNLKQNDQ PMDILQIINC LTTIYDRLEQ 1080EHNNLVNVPL CVDMCLNWLL NVYDTGRTGR IRVLSFKTGI ISLCKAHLED KYRYLFKQVA 1140SSTGFCDQRR LGLLLHDSIQ IPRQLGEVAS FGGSNIEPSV RSCFQFANNK PEIEAALFLD 1200WMRLEPQSMV WLPVLHRVAA AETAKHQAKC NICKECPIIG FRYRSLKHFN YDICQSCFFS 1260GRVAKGHKMH YPMVEYCTPT TSGEDVRDFA KVLKNKFRTK RYFAKHPRMG YLPVQTVLEG 1320DNMETPDTM 1329 SEQ ID DNA sequence of human mini-dystrophin gene 43990NO: 28 atggtttggt gggaagaagt agaggactgt tatgaaagag aagatgttca aaagaaaaca60 ttcacaaaat gggtaaatgc acaattttct aagtttggga agcagcatat tgagaacctc 120ttcagtgacc tacaggatgg gaggcgcctc ctagacctcc tcgaaggcct gacagggcaa 180aaactgccaa aagaaaaagg atccacaaga gttcatgccc tgaacaatgt caacaaggca 240ctgcgggttt tgcagaacaa taatgttgat ttagtgaata ttggaagtac tgacatcgta 300gatggaaatc ataaactgac tottggtttg atttggaata taatcctcca ctggcaggtc 360aaaaatgtaa tgaaaaatat catggctgga ttgcaacaaa ccaacagtga aaagattctc 420ctgagctggg tccgacaatc aactcgtaat tatccacagg ttaatgtaat caacttcacc 480accagctggt ctgatggcct ggctttgaat gctctcatcc atagtcatag gccagaccta 540tttgactgga atagtgtggt ttgccagcag tcagccacac aacgactgga acatgcattc 600aacatcgcca gatatcaatt aggcatagag aaactactcg atcctgaaga tgttgatacc 660acctatccag ataagaagtc catcttaatg tacatcacat cactcttcca agttttgcct 720caacaagtga gcattgaagc catccaggaa gtggaaatgt tgccaaggcc acctaaagtg 780actaaagaag aacattttca gttacatcat caaatgcact attctcaaca gatcacggtc 840agtctagcac agggatatga gagaacttct tcccctaagc ctcgattcaa gagctatgcc 900tacacacagg ctgcttatgt caccacctct gaccctacac ggagcccatt tccttcacag 960catttggaag ctcctgaaga caagtcattt ggcagttcat tgatggagag tgaagtaaac 1020ctggaccgtt atcaaacagc tttagaagaa gtattatcgt ggcttctttc tgctgaggac 1080acattgcaag cacaaggaga gatttctaat gatgtggaag tggtgaaaga ccagtttcat 1140actcatgagg ggtacatgat ggatttgaca gcccatcagg gccgggttgg taatattcta 1200caattgggaa gtaagctgat tggaacagga aaattatcag aagatgaaga aactgaagta 1260caagagcaga tgaatctcct aaattcaaga tgggaatgcc tcagggtagc tagcatggaa 1320aaacaaagca atttacatag agttttaatg gatctccaga atcagaaact gaaagagttg 1380aatgactggc taacaaaaac agaagaaaga acaaggaaaa tggaggaaga gcctcttgga 1440cctgatcttg aagacctaaa acgccaagta caacaacata aggtgcttca agaagatcta 1500gaacaagaac aagtcagggt caattctctc actcacatgg tggtggtagt tgatgaatct 1560agtggagatc acgcaactgc tgctttggaa gaacaactta aggtattggg agatcgatgg 1620gcaaacatct gtagatggac agaagaccgc tgggttcttt tacaagacca gcctgaccta 1680gctcctggac tgaccactat tggagcctct cctactcaga ctgttactct ggtgacacaa 1740cctgtggtta ctaaggaaac tgccatctcc aaactagaaa tgccatcttc cttgatgttg 1800gaggtaccta ctcatagatt actgcaacag ttccccctgg acctggaaaa gtttcttgcc 1860tggcttacag aagctgaaac aactgccaat gtcctacagg atgctacccg taaggaaagg 1920ctcctagaag actccaaggg agtaaaagag ctgatgaaac aatggcaaga cctccaaggt 1980gaaattgaag ctcacacaga tgtttatcac aacctggatg aaaacagcca aaaaatcctg 2040agatccctgg aaggttccga tgatgcagtc ctgttacaaa gacgtttgga taacatgaac 2100ttcaagtgga gtgaacttcg gaaaaagtct ctcaacatta ggtcccattt ggaagccagt 2160tctgaccagt ggaagcgtct gcacctttct ctgcaggaac ttctggtgtg gctacagctg 2220aaagatgatg aattaagccg gcaggcacct attggaggcg actttccagc agttcagaag 2280cagaacgatg tacatagggc cttcaagagg gaattgaaaa ctaaagaacc tgtaatcatg 2340agtactcttg agactgtacg aatatttctg acagagcagc ctttggaagg actagagaaa 2400ctctaccagg agcccagaga gctgcctcct gaggagagag cccagaatgt cactcggctt 2460ctacgaaagc aggctgagga ggtcaatact gagtgggaaa aattgaacct gcactccgct 2520gactggcaga gaaaaataga tgagaccctt gaaagactcc aggaacttca agaggccacg 2580gatgagctgg acctcaagct gcgccaagct gaggtgatca agggatcctg gcagcccgtg 2640ggcgatctcc tcattgactc tctccaagat cacctcgaga aagtcaaggc acttcgagga 2700gaaattgcgc ctctgaaaga gaacgtgagc cacgtcaatg accttgctcg ccagcttacc 2760actttgggca ttcagctctc accgtataac ctcagcactc tggaagacct gaacaccaga 2820tggaagcttc tgcaggtggc cgtcgaggac cgagtcaggc agctgcatga agcccacagg 2880gactttggtc cagcatctca gcactttctt tccacgtctg tccagggtcc ctgggagaga 2940gccatctcgc caaacaaagt gccctactat atcaaccacg agactcaaac aacttgctgg 3000gaccatccca aaatgacaga gctctaccag tctttagctg acctgaataa tgtcagattc 3060tcagcttata ggactgccat gaaactccga agactgcaga aggccctttg cttggatctc 3120ttgagcctgt cagctgcatg tgatgccttg gaccagcaca acctcaagca aaatgaccag 3180cccatggata tcctgcagat tattaattgt ttgaccacta tttatgaccg cctggagcaa 3240gagcacaaca atttggtcaa cgtccctctc tgcgtggata tgtgtctgaa ctggctgctg 3300aatgtttatg atacgggacg aacagggagg atccgtgtcc tgtcttttaa aactggcatc 3360atttccctgt gtaaagcaca tttggaagac aagtacagat accttttcaa gcaagtggca 3420agttcaacag gattttgtga ccagcgcagg ctgggcctcc ttctgcatga ttctatccaa 3480attccaagac agttgggtga agttgcatcc tttgggggca gtaacattga gccaagtgtc 3540cggagctgct tccaatttgc taataataag ccagagatcg aagcggccct cttcctagac 3600tggatgagac tggaacccca gtccatggtg tggctgcccg tcctgcacag agtggctgct 3660gcagaaactg ccaagcatca ggccaaatgt aacatctgca aagagtgtcc aatcattgga 3720ttcaggtaca ggagtctaaa gcactttaat tatgacatct gccaaagctg ctttttttct 3780ggtcgagttg caaaaggcca taaaatgcac tatcccatgg tggaatattg cactccgact 3840acatcaggag aagatgttcg agactttgcc aaggtactaa aaaacaaatt tcgaaccaaa 3900aggtattttg cgaagcatcc ccgaatgggc tacctgccag tgcagactgt cttagagggg 3960gacaacatgg aaactcccga cacaatgtag 3990

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-93. (canceled)
 94. A recombinant AAV (rAAV) particle comprising anAAV9 capsid and a vector genome, said genome comprising a first AAV2inverted terminal repeat (ITR), a muscle-specific transcriptionalregulatory element operably linked to a nucleotide sequence encoding ahuman mini-dystrophin protein consisting of the amino acid sequence ofSEQ ID NO:7, a transcription termination sequence, and a second AAV2ITR.
 95. The rAAV particle of claim 94, wherein said vector genome issingle-stranded DNA.
 96. The rAAV particle of claim 94, wherein saidnucleotide sequence encoding human mini-dystrophin protein iscodon-optimized.
 97. The rAAV particle of claim 96, whereincodon-optimization increases GC content relative to wild-type codingsequence.
 98. The rAAV particle of claim 97, wherein the GC content ofsaid nucleotide sequence is at least 60%.
 99. The rAAV particle of claim96, wherein said nucleotide sequence encoding human mini-dystrophinprotein is human codon-optimized.
 100. The rAAV particle of claim 94,wherein said muscle-specific transcriptional regulatory element confersmuscle tissue-specific expression in skeletal and cardiac muscle. 101.The rAAV particle of claim 100, wherein said muscle-specifictranscriptional regulatory element is a synthetic enhancer and promoterderived from a muscle creatinine kinase gene.
 102. The rAAV particle ofclaim 96, wherein said nucleotide sequence encoding humanmini-dystrophin protein is at least 90% identical to the nucleotidesequence of SEQ ID NO:1.
 103. The rAAV particle of claim 102, whereinsaid muscle-specific transcriptional regulatory element comprises thenucleotide sequence of SEQ ID NO:16.
 104. The rAAV particle of claim103, wherein said transcription termination sequence comprises thenucleotide sequence of SEQ ID NO:17.
 105. The rAAV particle of claim104, wherein said vector genome comprises the nucleotide sequence of SEQID NO:18, or the reverse complement thereof.
 106. A method of making therAAV particle of claim 94, comprising: introducing into a cell apolynucleotide comprising the nucleotide sequence of said vector genome,a polynucleotide comprising an AAV rep gene, a polynucleotide comprisingan AAV9 cap gene and, optionally, a polynucleotide encoding one or moreviral helper genes, incubating said cell; and purifying the rAAVparticles produced thereby.
 107. A rAAV particle produced by the methodof claim 106.